EP4271364A1 - Mizellare nanopartikel und verwendungen davon - Google Patents

Mizellare nanopartikel und verwendungen davon

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Publication number
EP4271364A1
EP4271364A1 EP21914850.9A EP21914850A EP4271364A1 EP 4271364 A1 EP4271364 A1 EP 4271364A1 EP 21914850 A EP21914850 A EP 21914850A EP 4271364 A1 EP4271364 A1 EP 4271364A1
Authority
EP
European Patent Office
Prior art keywords
carrier unit
cationic carrier
payload
anionic
micelle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21914850.9A
Other languages
English (en)
French (fr)
Inventor
Jin-Hyeob RYU
Yu Na Lim
Hyun Su Min
Han Seok Koh
Dae Hoon Kim
Hyun-Jeong CHO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biorchestra Co Ltd
Original Assignee
Biorchestra Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biorchestra Co Ltd filed Critical Biorchestra Co Ltd
Publication of EP4271364A1 publication Critical patent/EP4271364A1/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Definitions

  • the present disclosure provides cationic carrier units and micelle systems, which can be used to deliver anionic payloads (e.g., RNA and/or DNA) across physiological permeation barriers, e.g., the brain blood barrier.
  • anionic payloads e.g., RNA and/or DNA
  • physiological permeation barriers e.g., the brain blood barrier.
  • BACKGROUND ART Intracellular drug delivery is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane.
  • the cell membrane is selectively permeable to non-polar therapeutic agents, which are lipid soluble and can pass through the cell membrane.
  • highly charged therapeutic agents such as mRNA are effectively excluded by the cell membrane.
  • Polynucleotides do not readily permeate the cellular membrane due to the charge repulsion between the negatively charged membrane and the high negative charge on the polynucleotide. As a result, polynucleotides have poor bioavailability and uptake into cells, typically less than 1% (Dheur et al, Nucleic Acid Drug Dev., 9:522 (1999); Park et al, J Controlled Release, 93:188 (2003)). Since most polynucleotides are generally above 5,000 Da, they cannot readily diffuse through cellular membranes and uptake into cells is limited primarily to pinocytotic or endocytotic processes.
  • polynucleotides can accumulate in lysosomal compartments, limiting their access to the cytoplasm or the nucleus.
  • Parenterally administered polynucleotides are also highly susceptible to rapid nuclease degradation both inside and outside the cytoplasm. Studies show rapid degradation of polynucleotides in blood after i.v. administration, with a half-life of about 30 minutes (Geary et al, J. Pharmacol. Exp. Ther.296:890-897 (2001)).
  • the problems facing the delivery of polynucleotide e.g., mRNA, can roughly be divided into two parts.
  • the therapeutic polynucleotide must be formulated in such a way that it can be delivered to the cytoplasm and second, the polynucleotide must reach the cell nucleus intact and fully functional.
  • nucleotides e.g., gene therapy
  • Efforts aimed at improving the transmembrane delivery of nucleic acids have utilized protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate-mediated transformation.
  • a cationic carrier unit comprising [CC]-L1-[CM]-L2-[HM] (Schema I); [CC]-L1-[HM]-L2-[CM] (Schema II); [HM]-L1-[CM]-L2-[CC] (Schema III); [HM]-L1-[CC]-L2-[CM] (Schema IV); [CM]-L1-[CC]-L2-[HM] (Schema V); or [CM]-L1-[HM]-L2-[CC] (Schema VI); wherein CC is a positively charged carrier moiety; CM is a crosslinking moiety; HM is a hydrophobic moiety; and, L1 and L2 are independently optional linkers, and wherein the number of HM is less than 40% relative to [CC] and [CM].
  • the number of HM is less than 39%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or about 1% relative to [CC] and [CM].
  • the number of HM is between about 35% and about 1%, about 35% and about 5%, about 35% and about 10%, about 35% and about 15%, about 35% and about 20%, about 35% and about 25%, about 35% and about 30%, about 30% and about 1%, about 30% and about 5%, about 30% and about 10%, about 30% and about 15%, about 30% and about 20%, about 30% and about 25%, about 25% and about 1%, about 25% and about 5%, about 25% and about 10%, about 25% and about 15%, about 25% and about 20%, about 20% and about 1%, about 20% and about 5%, about 20% and about 10%, about 20% and about 15%, about 15% and about 1%, about 15% and about 5%, about 15% and about 10%, about 10% and about 1%, or about 10% and about 5% relative to [CC] and [CM].
  • the number of HM is between about 39% and about 30%, about 30% and about 20%, about 20% and about 10%, about 10% and about 5%, and about 5% and about 1% relative to [CC] and [CM]. In some aspects, the number of HM is about 39%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% relative to [CC] and [CM]. In some aspects, the cationic carrier unit is capable of interacting with an anionic payload.
  • the anionic payload comprises a nucleotide sequence having less than 4000 nucleotides, less than about 3500, less than about 3000, less than about 2500, less than about 2000, less than about 1500, less than about 1000, less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 200, or less than about 150 in length.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 1 and about 20, about 1 and about 19, about 1 and about 18, about 1 and about 17, about 1 and about 16, , about 1 and about 15, about 1 and about 14, about 1 and about 13, about 1 and about 12, about 1 and about 11, about 1 and about 10, about 1 and about 9, about 1 and about 8, about 1 and about 7, about 1 and about 6, or about 1 and about 5.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • the anionic payload comprises a nucleotide sequence having about 100 nucleotides to about 1000 nucleotides in length.
  • the number of HM is between 39% and about 30%, about 30% and about 20%, about 20% and about 10%, about 10% and about 5%, and about 5% and about 1% relative to [CC] and [CM].
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 1 and about 10 or about 3 and about 7. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 1 and about 2, about 2 and about 3, about 3 and about 4, or about 4 and about 5.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 1, about 2, about 3, about 4, or about 5.
  • the anionic payload comprises a nucleotide sequence having about 1000 nucleotides to about 2000 nucleotides in length.
  • the number of HM is less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% relative to [CC] and [CM].
  • the number of HM is between about 30% and about 20%, about 30% and about 25%, about 25% and about 20%, about 25% and about 15%, about 20% and about 10%, about 20% and about 5%, about 10% and about 1%, about 10% and about 5%, and about 5% and about 1% relative to [CC] and [CM].
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 4 and about 7.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 4 and about 5 or about 5 and about 6. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 4, about 5, about 6, or about 7. [0013] In some aspects, the anionic payload comprises a nucleotide sequence having about 2000 nucleotides to about 3000 nucleotides in length.
  • the number of HM is less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% relative to [CC] and [CM].
  • the number of HM is less than about 20% to about 1%, from about 20% to about 5%, from about 20% to about 10%, from about 20% to about 15%, from about 15% to about 1%, from about 15% to about 5%, from about 15% to 10%, from about 10% to about 1%, from about 10% to about 5%, or from about 5% to about 1% nucleotides relative to [CC] and [CM].
  • the number of HM is about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% relative to [CC] and [CM].
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 6 and about 9.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 6 and about 7 or about 7 and about 8. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 6, about 7, about 8, or about 9. [0014] In some aspects, the anionic payload comprises a nucleotide sequence having about 3000 nucleotides to about 4000 nucleotides in length.
  • the number of HM is less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% relative to [CC] and [CM]. In some aspects, the number of HM is from about 10% to about 1%, from about 10% to about 5%, or from about 5% to about 1% nucleotides relative to [CC] and [CM]. In some aspects, the number of HM is about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% relative to [CC] and [CM].
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 7 and about 10. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 7 and about 8 or about 8 and about 9.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 7, about 8, about 9, or about 10.
  • the anionic payload comprises mRNA, cDNA, or any combination thereof.
  • the cationic carrier further comprises a water-soluble polymer (WP).
  • WP water-soluble polymer
  • the water-soluble polymer is attached to [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the N terminus of [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the C terminus of [CC], [HM], or [CM].
  • the carrier unit comprises: [WP]-L3]-[CC]-L1-[CM]-L2-[HM] (Schema I’); [WP]-L3]-[CC]-L1-[HM]-L2-[CM] (Schema II’); [WP]-L3]-[HM]-L1-[CM]-L2-[CC] (Schema III’); [WP]-L3]-[HM]-L1-[CC]-L2-[CM] (Schema IV’); [WP]-L3]-[CM]-L1-[CC]-L2-[HM] (Schema V’); or [WP]-L3]-[CM]-L1-[HM]-L2-[CC] (Schema VI’).
  • the water-soluble polymer comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines ("POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG").
  • the water-soluble polymer comprises: , wherein n is 1-1000. [0019] In some aspects, n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141.
  • n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 140 to about 150, or about 150 to about 160.
  • the water-soluble polymer is linear, branched, or dendritic.
  • the cationic carrier moiety comprises one or more amino acids.
  • the cationic carrier moiety comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at last 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 basic amino acids, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about
  • the cationic carrier moiety comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, or at least about 150 amino acids.
  • the cationic carrier moiety comprises about 10 to about 60, about 15 to about 60, about 20 to about 60, about 25 to about 60, about 30 to about 60, about 35 to about 60, about 40 to about 60, about 10 to about 55, about 15 to about 55, about 20 to about 55, about 25 to about 55, about 30 to about 55, about 35 to about 55, about 40 to about 55, about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 50, about 35 to about 50, about 40 to about 50, about 10 to about 45, about 15 to about 45, about 20 to about 45, about 25 to about 45, about 30 to about 45, about 35 to about 45, about 40 to about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 35 to about 40, about 35 to about 40, about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 35 to
  • the cationic carrier moiety comprises about 10, about 20, about 30, about 40, about 50, or about 60 amino acids.
  • the amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the cationic carrier moiety comprises about 20, about 30, about 40, about 50, or about 60 lysines.
  • the cationic carrier moiety comprises about 40 lysine monomers.
  • the crosslinking moiety comprises one or more amino acids linked to a crosslinking agent.
  • the crosslinking agent comprises a thiol group, a thiol derivative, or any combination thereof.
  • the crosslinking agent comprises a thiol group.
  • the amino acids in the crosslinking moiety comprise at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 amino acids.
  • the amino acids in the crosslinking moiety comprise about 1 to about 40, about 5 to about 40, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 1 to about 35, about 5 to about 35, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 10 to about 50, about 15 to about 50, about 20 to about 50, about 25 to about 50, about 30 to about 40, about 10 to about 45, about 15 to about 45, about 20 to about 45, about 25 to about 45, about 30 to about 45, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, or about 30 to about 40 amino acids.
  • the amino acids in the crosslinking moiety comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids. In some aspects, the amino acids in the crosslinking moiety comprise arginine, lysine, histidine, or any combination thereof. In some aspects, the amino acids in the crosslinking moiety comprise about 35 lysines. In some aspects, the amino acids in the crosslinking moiety comprise about 23 lysines. In some aspects, wherein the amino acids in the crosslinking moiety comprises about 16 lysines. [0023] In some aspects, the hydrophobic moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue microenvironment.
  • the hydrophobic moiety is capable of modulating an immune response. In some aspects, the hydrophobic moiety is capable of modulating a tumor microenvironment in a subject with a tumor. [0024] In some aspects, the hydrophobic moiety is capable of inhibiting or reducing hypoxia in the tumor microenvironment. In some aspects, the hydrophobic moiety comprises one or more amino acids linked to an imidazole derivative, an amino acid, a vitamin, or any combination thereof. [0025] In some aspects, the hydrophobic moiety is capable of inhibiting or reducing an inflammatory response. In some aspects, the hydrophobic moiety is one or more amino acids linked to a vitamin.
  • the vitamin comprises a cyclic ring or cyclic heteroatom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: wherein each of Y 1 and Y 2 are independently selected from C, N, O, and S, and wherein n is 1 or 2.
  • the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any combination thereof.
  • the vitamin is vitamin B3.
  • the hydrophobic moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, or at least 32 amino acids, each linked to a vitamin.
  • the hydrophobic moiety comprises from about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 5 to about 35, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, about 5 to about 10, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 20, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 30, or about 25 to about 30 amino acids, each linked to a vitamin.
  • the hydrophobic moiety comprises the hydrophobic moiety comprises about 2 vitamin B3, about 3 vitamin B3, about 4 vitamin B3, about 5 vitamin B3, about 6 vitamin B3, about 7 vitamin B3, about 8 vitamin B3, about 9 vitamin B3, about 10 vitamin B3, about 11 vitamin B3, about 12 vitamin B3, about 13 vitamin B3, about 14 vitamin B3, about 15 vitamin B3, about 16 vitamin B3, about 17 vitamin B3, about 18 vitamin B3, about 19 vitamin B3, about 20 vitamin B3, about 21 vitamin B3, about 22 vitamin B3, about 23 vitamin B3, about 24 vitamin B3, about 25 vitamin B3, about 26 vitamin B3, about 27 vitamin B3, about 28 vitamin B3, about 29 vitamin B3, about 30, about 31, about 32, about 33, about 34, or about 35 amino acids, each linked to vitamin B3.
  • the cationic carrier unit comprises about 35 to about 45 lysines
  • the crosslinking moiety comprises about 20 to about 40 lysine-thiol
  • the hydrophobic moiety comprises about 1 to about 10 lysine-vitamin B3.
  • the cationic carrier moiety comprises about 35 to about 45 lysines
  • the crosslinking moiety comprises about 10 to about 20 lysine-thiol
  • the hydrophobic moiety comprises about 1 to about 10 lysine-vitamin B3.
  • the cationic carrier moiety comprises about 35 to about 45 lysines
  • the crosslinking moiety comprises about 10 to about 30 lysine-thiol
  • the hydrophobic moiety comprises about 1 to about 10 lysine-vitamin B3.
  • the cationic carrier moiety comprises about 35 to about 45 lysines
  • the crosslinking moiety comprises about 13 to about 25 lysine-thiol
  • the hydrophobic moiety comprises about 1 to about 20 lysine-vitamin B3.
  • the cationic carrier moiety comprises about 35 to about 45 lysines, the crosslinking moiety comprises about 13 to about 25 lysine-thiol, and the hydrophobic moiety comprises about 1 to about 20 lysine-vitamin B3.
  • the cationic carrier unit comprises a water-soluble biopolymer moiety comprising about 120 to about 130 PEG units.
  • the cationic carrier unit further comprises a targeting moiety (TM).
  • the targeting moiety is capable of targeting a tissue.
  • the targeting moiety is capable of being transported by large neutral amino acid transporter 1 (LAT1).
  • LAT1 large neutral amino acid transporter 1
  • the targeting moiety is an amino acid.
  • the targeting moiety comprises a branched-chain or aromatic amino acid.
  • the targeting moiety is phenylalanine, valine, leucine, and/or isoleucineIn some aspects, the amino acid is phenylalanine.
  • the targeting moiety is linked to the water-soluble polymer. In some aspects, the targeting moiety is linked to the water- soluble polymer by a linker.
  • the present disclosure also provides a micelle comprising the cationic carrier unit disclosed herein and an anionic payload, wherein the cationic carrier moiety of the cationic carrier complex and the anionic payload are associated with each other.
  • the association is a covalent bond.
  • the association is a non-covalent bond.
  • the association is an ionic bond.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together and wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 1 and about 20.
  • the N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.
  • the positive charge of the cationic carrier moiety of the cationic carrier unit is sufficient to form a micelle when mixed with an anionic payload in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 3, about 4, about 5, about 6, about 7, about 8, or about 9.
  • the cationic carrier unit is capable of protecting the anionic payload from degradation by a DNase and/or an RNase.
  • the anionic payload is not conjugated to the cationic carrier unit by a covalent bond and/or the anionic payload interacts with the cationic carrier moiety of the cationic carrier unit only via an ionic interaction.
  • the half-life of the anionic payload is extended compared to the half-life of a free anionic payload not incorporated into a micelle.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 4 and about 7. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 4 and about 5 or about 5 and about 6. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 4, about 5, about 6, or about 7.
  • the anionic payload that can be mixed with a cationic carrier unit to form a micelle comprises a nucleotide sequence having about 2000 nucleotides to about 3000 nucleotides in length.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, and wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 6 and about 9.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 6 and about 7 or about 7 and about 8.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the the N/P ratio of the cationic carrier unit and the anionic payload is about 6, about 7, about 8, or about 9.
  • the anionic payload that can be mixed with a cationic carrier unit to form a micelle comprises a nucleotide sequence having about 3000 nucleotides to about 4000 nucleotides in length.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 7 and about 10.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 7 and about 8 or about 8 and about 9. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 7, about 8, about 9, or about 10.
  • the diameter of the micelle is between about 10 nm and about 200 nm, between about 20 nm and about 200 nm, between about 1nm and 100nm, between about 10nm and about 100nm, between about 10nm and about 90nm, between about 10nm and about 80nm, between about 10nm and about 70nm, between about 20nm and about 100nm, between about 20nm and about 90nm, between about 20nm and about 80nm, between about 20nm and about 70nm, between about 30nm and about 100nm, between about 30nm and about 90nm, between about 30nm and about 80nm, between about 30nm and about 70nm, between about 40nm and about 100nm, between about 40nm and about 90nm, between about 40nm and about 80nm, or between about 40nm and about 70nm.
  • the anionic payload comprises a nucleic acid.
  • the nucleic acid comprises mRNA, miRNA sponge, tough decoy miRNA, cDNA, pDNA, PNA, BNA, (ASO), aptamer, or any combination thereof.
  • the nucleic acid comprises at least one nucleoside analog.
  • the nucleoside analog comprises Locked Nucleic Acid (LNA); 2'-0-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro- ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-0-methyl nucleic acid (2'-OMe), 2'-0- methoxyethyl nucleic acid (2'-MOE), or any combination thereof.
  • LNA Locked Nucleic Acid
  • ANA arabino nucleic acid
  • INA intercalating nucleic acid
  • cEt constrained ethyl nucleoside
  • 2'-OMe 2'-0-methyl nucleic acid
  • 2'-MOE methoxyethyl nucleic acid
  • the nucleic acid comprises a nucleotide sequence having at least about 100, at least about 500, at least about 1000, at least about 1500, at least about 2000, at least about 2500, at least about 3000, at least about 3500, or at least about 4000 nucleotides in length.
  • the nucleotide sequence has a backbone, which comprises a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoramidate linkage, a phosphorothioate linkage, and combinations thereof.
  • the cationic carrier unit further comprises a targeting moiety, which is linked to the water-soluble polymer optionally via a linker.
  • the present disclosure also provides a composition comprising the cationic carrier unit disclosed herein and an anionic payload. Also provided is a pharmaceutical composition comprising a cationic carrier unit, composition, or micelle disclosed herein, and a pharmaceutically acceptable carrier. [0041] The present disclosure also provides a method of preparing the cationic carrier unit disclosed herein comprising linking the cationic carrier moiety to the crosslinking moiety and the hydrophobic moiety. In some aspects, the method further comprises linking a water-soluble polymer and a targeting moiety. In some aspects, the method of preparing a micelle disclosed herein comprises mixing the cationic carrier unit with the anionic payload in solution. In some aspects, the method further comprises purifying the micelle.
  • the present disclosure also provides a method of treating a disease or condition in a subject in need thereof comprising administering a micelle or pharmaceutical composition of the present disclosure to the subject.
  • the anionic payload in the core of the micelle exhibits a longer half-life than a corresponding anionic payload not integrated into a micelle.
  • the subject is a mammal.
  • FIGs. 1A-1C show exemplary architectures of carrier units and micelles of the present disclosure.
  • the exemplary carrier units comprise an optional tissue-specific targeting moiety, water-soluble polymer, and cationic carrier unit (which can, respectively, interact with anionic payloads) (FIG. 1A).
  • the cationic carrier and anionic payload are not tethered and interact electrostatically.
  • FIG. 1B shows a schematic diagram of an anionic payload.
  • the cationic carrier and anionic payload are tethered and interact electrostatically.
  • FIG.1C shows a schematic diagram of a micelle comprising a cationic carrier and anionic payload of FIGs.1A and 1B.
  • FIGs. 2A-2E show exemplary compositions of cationic carrier units.
  • FIG 2A shows a carrier unit comprising a targeting moiety, water-soluble polymer (e.g., polyethylene glycol), and a cationic carrier unit comprising 80 lysine residues, wherein 64 lysine residues are unmodifided (e.g., contain positively charged –NH3+) and wherein 16 lysine residues are modified for crosslinking (e.g., linked to a thiol, alkyl thiol, or lysine-thiol).
  • a targeting moiety e.g., water-soluble polymer
  • a cationic carrier unit comprising 80 lysine residues, wherein 64 lysine residues are unmodifided (e.g., contain positively charged –NH3+) and wherein 16 lysine residues are modified for crosslinking (e.g., linked to a thiol, alkyl thiol, or lysine-thiol).
  • FIG. 2B shows a carrier unit comprising a targeting moiety, water soluble polymer (e.g., polyethylene glycol), and a cationic carrier unit comprising 80 lysine residues, wherein 40 lysine residues are unmodifided (e.g., contain a positively charged amine group, e.g., -NH3+) and wherein 35 lysine residues are modified for crosslinking (e.g., linked to a thiol, alkyl thiol, or lysine-thiol) and wherein 5 lysine residues are modified to contain a hydrophobic moiety (e.g., a vitamin).
  • 2C shows a carrier unit comprising a targeting moiety, water soluble polymer (e.g., polyethylene glycol), and a cationic carrier unit comprising 80 lysine residues, wherein 38 lysine residues are unmodifided (e.g., contain a positively charged amine group, e.g., -NH3+) and wherein 23 lysine residues are modified for crosslinking (e.g., linked to a thiol, alkyl thiol, or lysine-thiol) and wherein 19 lysine residues are modified to contain a hydrophobic moiety (e.g., a vitamin).
  • a hydrophobic moiety e.g., a vitamin
  • 2D shows a carrier unit comprising a targeting moiety, water soluble polymer (e.g., polyethylene glycol), and a cationic carrier unit comprising 80 lysine residues, wherein 32 lysine residues are unmodifided (e.g., contain a positively charged amine group, e.g., -NH3+) and wherein 16 lysine residues are modified for crosslinking (e.g., linked to a thiol, alkyl thiol, or lysine-thiol) and wherein 32 lysine residues are modified to contain a hydrophobic moiety (e.g., a vitamin).
  • FIG. 2E shows a carrier unit comprising a targeting moiety, water-soluble polymer (e.g., polyethylene glycol), and a cationic carrier unit comprising 80 lysine residues, wherein 63 lysine residues are unmodifided (e.g., contain a positively charged amine group, e.g., -NH3+) and wherein 17 lysine residues are modified to contain a hydrophobic moiety (e.g., a vitamin).
  • FIG. 3 shows a tissue specific targeting polymer structure for nucleotide micelle deleivery and 1 H-NMR characteristics of a carrier unit.
  • FIG. 4A-4D show particle size and count rate at increasing N/P ratios for Compound A and nucleotide micelles measured by Zeta-sizer.
  • FIG. 4A shows a schematic representation of Compound A (FIG.2A).
  • FIG.4B-4D shows a the count rate and size of micelles comprising 800 nucleotides, 1,800 nucleotides, and 3,800 nucleotides, respectively, (e.g., anionic payload) and Compound A as the cationic carrier unit from an N/P ratio of 1-10.
  • FIGs. 5A-5D show particle size and count rate at increasing N/P ratios for Compound B and nucleotide micelles measured by Zeta-sizer.
  • FIG. 5A shows a schematic representation of Compound B (FIG.2B).
  • FIG.5B-5D shows a the count rate and size of micelles comprising 800 nucleotides, 1,800 nucleotides, and 3,800 nucleotides, respectively, (e.g., anionic payload) and Compound A as the cationic carrier unit from an N/P ratio of 1-10.
  • FIGs. 6A-6D show particle size and count rate at increasing N/P ratios for Compound C and nucleotide micelles measured by Zeta-sizer.
  • FIG. 6A shows a schematic representation of Compound C (FIG.2C).
  • FIG.6B-6D shows a the count rate and size of micelles comprising 800 nucleotides, 1,800 nucleotides, and 3,800 nucleotides, respectively, (e.g., anionic payload) and Compound A as the cationic carrier unit from an N/P ratio of 1-10.
  • FIGs. 7A-7D show particle size and count rate at increasing N/P ratios for Compound D and nucleotide micelles measured by Zeta-sizer.
  • FIG. 7A shows a schematic representation of Compound D (FIG.2D).
  • FIG.7B-7D shows a the count rate and size of micelles comprising 800 nucleotides, 1,800 nucleotides, and 3,800 nucleotides, respectively, (e.g., anionic payload) and Compound D as the cationic carrier unit from an N/P ratio of 1-10.
  • FIGs. 8A-8D show particle size and count rate at increasing N/P ratios for Compound E and nucleotide micelles measured by Zeta-sizer.
  • FIG. 8A shows a schematic representation of Compound E (FIG.2E).
  • FIG.8B-8D shows a the count rate and size of micelles comprising 800 nucleotides, 1,800 nucleotides, and 3,800 nucleotides, respectively, (e.g., anionic payload) and Compound E as the cationic carrier unit from an N/P ratio of 1-10.
  • FIGs. 9A-9B show particle size of mRNA micelle after micelle formulation between polymer (Compounds B, C, D) and mRNA (800 nt, 1800 nt, 3800 nt) individually at optimized N/P ratios (FIG.9A) and fixed N/P ratios (FIG.9B).
  • FIGs. 9A-9B show particle size of mRNA micelle after micelle formulation between polymer (Compounds B, C, D) and mRNA (800 nt, 1800 nt, 3800 nt) individually at optimized N/P ratios (FIG.9A) and fixed N/P ratios (FIG.9B).
  • FIG. 10A-10C show particle size distribution of mRNA containing micelles prepared with compound B and mRNA nucleotide lengths of 800 (FIG. 10A), 1,800 (FIG. 10B), and 3,800 (FIG.10C).
  • FIG. 11 shows relative mRNA expression level after transfection of PBS, mRNA encapsulated micelle and Lipofectamine 2000 with mRNA in HEK 293T cells. 5 ⁇ g of mRNA formulated with PBS, polymeric carrier or Lipofectamine 2000 were transfected into the HEK- 293T cells for 30 min. The mRNA level of gene was normalized on the basis of hGAPDH gene expression. The relative mRNA expression levels of the gene were calculated using the 2- ⁇ Ct method.
  • FIGs. 12A-12D show the protein expression levels of LAT1 in the muscles of BALB/c and DBA/2J mice.
  • Western blot assays were performed to measure the LAT1 expression in BALB/c (FIG. 12A) and DBA/2J mice (FIG. 12B).
  • Relative expression levels of LAT1 in BALB/c (FIG.12C) and DBA/2J mice (FIG.12D) were quantified, and expression levels of LAT1 were normalized with respect to GAPDH levels.
  • FIGs.13A-13B show bioluminescence images of mice after administration of Luc- mRNA encapsulated in polymeric micelles (FIG.13A) or Lipofectamine (FIG.13B), respectively. The luminescence images were taken up to 8 days after intramuscular injection.
  • carrier units comprising a water-soluble biopolymer moiety (e.g., PEG), a charged moiety (e.g., a polylysine), a crosslinking moiety, and a hydrophobic moiety.
  • the cationic carrier units can be packaged into micelles when the units interact with anionic paylods, wherein the payload is located in the core of the micelle and the water-soluble biopolymer moiety is facing the solvent, and wherein the crosslinking moiety crosslinks one unit to the other carrier units.
  • anionic paylods wherein the payload is located in the core of the micelle and the water-soluble biopolymer moiety is facing the solvent, and wherein the crosslinking moiety crosslinks one unit to the other carrier units.
  • ranges recited are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints.
  • a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission. Accordingly, ‘a’ represents adenine, ‘c’ represents cytosine, ‘g’ represents guanine, ‘t’ represents thymine, and ‘u’ represents uracil. [0068] Amino acid sequences are written left to right in amino to carboxy orientation. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).
  • administration refers to introducing a composition, such as a micelle of the present disclosure, into a subject via a pharmaceutically acceptable route.
  • a composition such as a micelle of the present disclosure
  • introduction of a composition is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intra-arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly or topically.
  • Administration includes self-administration and the administration by another.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be “completely conserved” or “identical” if they are 100% identical to one another.
  • two or more sequences are said to be "highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some aspects, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. In some aspects, two or more sequences are said to be "conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another.
  • two or more sequences are said to be "conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of a polynucleotide or polypeptide or may apply to a portion, region or feature thereof.
  • the term "derived from,” as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.
  • a nucleic acid sequence that is derived from a second nucleic acid sequence can include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.
  • the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis.
  • the mutagenesis used to derive nucleotides or polypeptides can be intentionally directed or intentionally random, or a mixture of each.
  • the mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from the first can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived nucleotide or polypeptide can be made by appropriate screening methods, e.g., as discussed herein.
  • Mutagenesis of a polypeptide typically entails manipulation of the polynucleotide that encodes the polypeptide.
  • a nucleotide or amino acid sequence that is derived from a second nucleotide or amino acid sequence has a sequence identity of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 8
  • complementary and complementarity refer to two or more oligomers (i.e., each comprising a nucleobase sequence), or between an oligomer and a target gene, that are related with one another by Watson-Crick base-pairing rules.
  • nucleobase sequence “T-G-A (5’ ⁇ 3’) is complementary to the nucleobase sequence “A-C-T (3’ ⁇ 5’).
  • Complementarity may be "partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
  • complementarity between a given nucleobase sequence and the other nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%.
  • the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
  • downstream nucleotide sequences relate to sequences that follow the starting point of transcription.
  • the translation initiation codon of a gene is located downstream of the start site of transcription.
  • excipient and “carrier” are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Generally, the term “homology” implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both identity and similarity.
  • polymeric molecules are considered to be "homologous" to one another if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions).
  • the term "homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • identity refers to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules (e.g. DNA molecules and/or RNA molecules).
  • polypeptide molecules or polynucleotide molecules e.g. DNA molecules and/or RNA molecules.
  • identity without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity).
  • the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence.
  • the amino acids at corresponding amino acid positions, or bases in the case of polynucleotides are then compared. [0082] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences.
  • One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.
  • MAFFT MAFFT
  • ClustalW Clustal W
  • Clustal X or Clustal Omega MUSCLE
  • Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent of sequence identity value is rounded to the nearest tenth.
  • 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.
  • isolating or purifying as used herein is the process of removing, partially removing (e.g., a fraction) of a composition of the present disclosure from a sample containing contaminants.
  • an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount.
  • an isolated composition has an amount and/or concentration of desired composition of the present disclosure, at or above an acceptable amount and/or concentration and/or activity.
  • the isolated composition is enriched as compared to the starting material from which the composition is obtained. This enrichment can be by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.
  • isolated preparations are substantially free of residual biological products.
  • the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • the term "linked” as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively.
  • the first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence.
  • the term "linked” means not only a fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5’-end or the 3’-end, but also includes insertion of the whole first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively).
  • the first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or a linker.
  • the linker can be, e.g., a polynucleotide.
  • mismatch refers to one or more nucleobases (whether contiguous or separate) in an oligomer nucleobase sequence that are not matched to a target pre- mRNA according to base pairing rules. While perfect complementarity is often desired, some aspects can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target pre-mRNA. Variations at any location within the oligomer are included. In certain aspects, antisense oligomers of the disclosure include variations in nucleobase sequence near the termini, variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 subunits of the 5' and/or 3' terminus.
  • one, two, or three nucleobases can be removed and still provide on-target binding.
  • the terms “modulate,” “modify,” and grammatical variants thereof generally refer when applied to a specific concentration, level, expression, function or behavior, to the ability to alter, by increasing or decreasing, e.g., directly or indirectly promoting/stimulating/up-regulating or interfering with/inhibiting/down-regulating the specific concentration, level, expression, function or behavior, such as, e.g., to act as an antagonist or agonist.
  • a modulator can increase and/or decrease a certain concentration, level, activity or function relative to a control, or relative to the average level of activity that would generally be expected or relative to a control level of activity.
  • Nucleic acid “nucleic acid molecule,” “nucleotide sequence,” “polynucleotide,” and grammatical variants thereof are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA, and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
  • a "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • pharmaceutically-acceptable carrier “pharmaceutically-acceptable excipient,” and grammatical variations thereof, encompass any of the agents approved by a regulatory agency of the U.S.
  • the term "pharmaceutical composition” refers to one or more of the compounds described herein, such as, e.g., a micelle of the present disclosure, mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.
  • polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • DNA triple-, double- and single-stranded deoxyribonucleic acid
  • RNA triple-, double- and single-stranded ribonucleic acid
  • It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • a polynucleotide can be, e.g., an RNA, e.g., mRNA, or DNA.
  • the RNA is a synthetic RNA.
  • the synthetic RNA comprises at least one unnatural nucleobase.
  • all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine).
  • the terms "polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.
  • polypeptide refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly, disulfide linkages are found in multichain polypeptides.
  • polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • a "peptide" can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • Any amino acids, e.g., lysines, used in the context of the cationic carrier possess a positive charge by having its natural side group (e.g., -NH 3 + for lysine) or by having a modified side group.
  • any amino acids, e.g., lysines, used in the context of the crosslinking moiety or the hydrophobic moiety may not possess any positive charges and can be linked to a crosslinking agent (e.g., thiol) or a hydrophobic agent (e.g., vitamin B3), respectively, by an amide bond or a linker.
  • a crosslinking agent e.g., thiol
  • a hydrophobic agent e.g., vitamin B3
  • prevent refers partially or completely delaying onset of an disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • prophylactic refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • a “prophylaxis” refers to a measure taken to maintain health and prevent or delay the onset of a bleeding episode, or to prevent or delay symptoms associated with a disease or condition.
  • similarity refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. It is understood that percentage of similarity is contingent on the comparison scale used, i.e., whether the amino acids are compared, e.g., according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.
  • subject refers to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • domestic animals e.g., dogs, cats and the like
  • farm animals e.g., cows, sheep, pigs, horses and the like
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like
  • the phrase "subject in need thereof” includes subjects, such as mammalian subjects, that would benefit from administration of a micelle of the disclosure, e.g., to improve hemostasis.
  • the phrases "systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • terapéuticaally effective amount is the amount of reagent or pharmaceutical compound comprising a micelle of the present disclosure that is sufficient to a produce a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof.
  • a therapeutically effective amount can be a "prophylactically effective amount” as prophylaxis can be considered therapy.
  • treat refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also include prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • the term “treating” or “treatment” means inducing an immune response in a subject against an antigen.
  • upstream refers to a nucleotide sequence that is located 5’ to a reference nucleotide sequence. II.
  • Carrier Units [0112] The present disclosure provides carrier units that can self-assemble into micelles or be incorporated into micelles.
  • Carrier units of the present disclosure comprise a water-soluble biopolymer moiety (e.g., PEG), a charged carrier moiety, a crosslinking moiety, and a hydrophobic moiety.
  • the charged carrier moiety is cationic (e.g., a polylysine), as exemplified in FIG. 1.
  • Carrier units of the present disclosure can be used to deliver negatively payloads (e.g., therapeutic or diagnostic agents).
  • negatively payloads that can be delivered by micelles comprises at least about 100, at least about 1000, at least about 2000, at elast about 3000, or at least about 4000 nucleotides in length.
  • Carrier units with a cationic charged carrier moiety can be used for the delivery of anionic payloads, e.g., polynucleotides.
  • Carrier units with an anionic charged carrier moiety can be used for the delivery of cationic payloads, e.g., positively charged small molecule drugs. See FIG. 1.
  • the cationic charged carrier moiety and the anionic payloads can electrostatically interact with each other.
  • the resulting carrier unit:payload complex can have a "head” comprising the water- soluble biopolymer moiety and a "tail” comprising the cationic carrier moiety electrostatically bound to the payload.
  • Carrier unit:payload complexes can self-associate, alone or in combination with other molecules, to yield micelles in which the anionic payload is located in the core of the micelle and the water-soluble biopolymer moiety is facing the solvent.
  • micelles of the present disclosure encompasses not only classic micelles but also small particles, small micelles, micelles, rod-like structures, or polymersomes.
  • the carrier units of the present disclosure can also comprise a targeting moiety (e.g., a targeting ligand) covalently linked to the water-soluble biopolymer moiety via one or more optional linkers.
  • a targeting moiety e.g., a targeting ligand
  • the targeting moiety can be located on the surface of the micelle and can deliver the micelle to a specific target tissue, to a specific cell type, and/or facilitate transport across a physiological barrier (e.g., cell plasma membrane).
  • the micelles of the present disclosure can comprises more than one type of targeting moiety.
  • the carrier units of the present disclosure can also comprise moiety hydrophobic moiety (HM) covalently linked to the charged cationic carrier moiety.
  • HM hydrophobic moiety
  • the hydrophobic moiety can have, e.g., a therapeutic, a co-therapeutic effect, or positively affect the homeostasis of the target cell or target tissue.
  • the HM comprises one or more amino acids.
  • the HM comprises one or more amino acids linked to a hydrophobic molecule (e.g., a vitamin).
  • the Hm comprises one or more lysine residues covalently bound to a hydrophobic molecule (e.g., a vitamin).
  • a hydrophobic molecule e.g., a vitamin
  • the anionic payload is not covalently linked to the carrier unit.
  • the anionic payload can be covalently linked to the cationic carrier unit, e.g., a linker such as cleavable linker.
  • Non-limiting examples of various aspects are shown in the present disclosure. The disclosure refers in particular to the use of cationic carrier units, e.g., to deliver anionic payloads such as nucleic acids.
  • the disclosures can be equally applied to the delivery of cationic payloads or to the delivery of neutral payloads by reversing the charges of the carrier moiety and payload (i.e., using an anionic carrier moiety in the carrier unit to deliver a cationic payload), or by using a neutral payload linked to a cationic or anionic adapter that would electrostatically interact with an anionic or cationic carrier moiety, respectively.
  • the present disclosure provides cationic carrier units of Schema I through Schema VI [CC]-L1-[CM]-L2-[HM] (Schema I); [CC]-L1-[HM]-L2-[CM] (Schema II); [HM]-L1-[CM]-L2-[CC] (Schema III); [HM]-L1-[CC]-L2-[CM] (Schema IV); [CM]-L1-[CC]-L2-[HM] (Schema V); or [CM]-L1-[HM]-L2-[CC] (Schema VI); wherein CC is a cationic carrier moiety, e.g., a polylysine; CM is a crosslinking moiety; HM is a hydrophobic moiety, e.g., vitamin, e.g., vitamin B3; and, L1 and L2 are independently optional linkers.
  • CC is a cationic carrier moiety, e.g., a
  • the cationic carrier unit further comprises a water-soluble polymer (WP).
  • WP water-soluble polymer
  • the water-soluble polymer is attached to [CC], [HM], and/or [CM].
  • the water-soluble polymer is attached to the N terminus of [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the N terminus of [CC].
  • the water-soluble polymer is attached to the C terminus of [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the C terminus of [CC].
  • the cationic carrier unit comprises: [WP]-L3-[CC]-L1-[CM]-L2-[HM] (Schema I’); [WP]-L3-[CC]-L1-[HM]-L2-[CM] (Schema II’); [WP]-L3-[HM]-L1-[CM]-L2-[CC] (Schema III’); [WP]-L3-[HM]-L1-[CC]-L2-[CM] (Schema IV’); [WP]-L3-[CM]-L1-[CC]-L2-[HM] (Schema V’); or [WP]-L3-[CM]-L1-[HM]-L2-[CC] (Schema VI’).
  • the [WP] component can be connected to at least one targeting moiety, i.e., [T]n-[WP]-... wherein n is an integer, e.g., 1, 2 or 3.
  • FIGs. 2A-2E present a schematic representation of a cationic carrier unit of the present disclosure. For simplicity, the units in FIG. 2A-2E have been represented linearly.
  • the carrier units can comprises the CC, CM, and HM moieties organized in a branched scaffold arrangement (see FIG.2 and FIG.3), for example, with (i) a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol) attached to the N or C terminus of the CC moeity and (iii) a HM (e.g., lysine linked to a hydrophobic agent,e.g., lysine linked to Vitamin B3) attached to the N or C terminus of the CM moiety.
  • a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol
  • cationic carrier units of the present disclosure are mixed with an anionic payload (e.g., a nucleic acid) at an ionic ratio of about 20:about 1, i.e., the number of negative charges in the anionic payload is about 20 times higher than the number of positive charges in the cationic carrier moiety, to about 20:1, i.e., the number of positive charges in the cationic carrier moiety is about ten times higher than the number of negative charges in the anionic payload
  • the neutralization of negative charges in the anionic payload by positive charges in the cationic carrier moiety mainly via electrostatic interaction leads to the formation of a cationic carrier unit:anionic payload complex having an unaltered hydrophilic portion (comprising the WP moiety) and a substantially more hydrophobic portion (resulting from the association between the cationic carrier moiety plus hydrophobic moiety and the anionic payload).
  • the hydrophobic moiety can contribute its own positive charges to the positive charges of the cationic carrier moiety, which would interact with the negative charges of the anionic payload. It is to be understood that references to the interactions (e.g., electrostatic interactions) between a cationic carrier moiety and an anionic payload also encompass interactions between the charges of a cationic carrier moiety plus hydrophobic moiety and the charges of an anionic payload. [0127] The increase in the hydrophobicity of the cationic carrier moiety of the cationic carrier unit due to the neutralization of its positive charges via electrostatic interaction with the negative charges of the anionic payload results in an amphipathic complex.
  • Such amphipathic complexes can self-organize, alone or combination with other amphipathic components, into micelles.
  • the resulting micelles comprise the WP moieties facing the solvent (i.e., the WP moieties are facing the external surface of the micelle), whereas the CC and HM moieties as well as the associate payload (e.g., a nucleotide sequence, e.g., RNA, DNA, or any combination thereof) are in the core of the micelle.
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 1 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 1 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 5 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40, e.g., about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, e.g., 35 lysine-thio
  • PEG polyethylene glycol
  • the cationic carrier unit further comprises at least one targeting moiety attached to the WP moiety of the cationic carrier unit.
  • the number and/or density of targeting moieties displayed on the surface of the micelle can be modulated by using a specific ratio of cationic carrier units having targeting moieties to cationic carrier units not having targeting moieties.
  • the ratio of cationic carrier units having a targeting moiety to cationic carrier units not having a targeting moiety is at least about 1:5, at least about 1:10, at least about 1:20, at least about 1:30, at least about 1:40, at least about 1:50, at least about 1:60, at least about 1:70, at least about 1:80, at least about 1:90, at least about 1:100, at least about 1:120, at least about 1:140, at least about 1:160, at least about 1:180, at least about 1:200, at least about 1:250, at least about 1:300, at least about 1:350, at least about 1:400, at least about 1:450, at least about 1:500, at least about 1:600, at least about 1:700, at least about 1:800, at least about 1:900, or at least about 1:1000.
  • the cationic carrier unit comprises (i) a targeting moiety (A) which targets the transporter LAT1 (e.g., phenylalanine), (ii) a water soluble polymer which is PEG, (iii) a cationic carrier moiety comprising cationic polymer blocks which are lysine (iv) a crosslinking moiety comprising crosslinking polymer blocks which are lysines linked to crosslinking moieties, and (v) a hydrophobic moiety comprising hydrophobic polymer blocks which are lysines linked to vitamin B3.
  • a targeting moiety A
  • targets the transporter LAT1 e.g., phenylalanine
  • a water soluble polymer which is PEG e.g., a water soluble polymer which is PEG
  • a cationic carrier moiety comprising cationic polymer blocks which are lysine
  • iv a crosslinking moiety comprising crosslinking polymer blocks which are lys
  • a targeting moiety A
  • targets the transporter LAT1 e.g., phenylalanine
  • A targets the transporter LAT1 (e.g., phenylalanine)
  • the number (percentage) of HM is less than 39%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or about 1% relative to [CC] and [CM].
  • the number (percentage) of HM is between about 35% and about 1%, about 35% and about 5%, about 35% and about 10%, about 35% and about 15%, about 35% and about 20%, about 35% and about 25%, about 35% and about 30%, about 30% and about 1%, about 30% and about 5%, about 30% and about 10%, about 30% and about 15%, about 30% and about 20%, about 30% and about 25%, about 25% and about 1%, about 25% and about 5%, about 25% and about 10%, about 25% and about 15%, about 25% and about 20%, about 20% and about 1%, about 20% and about 5%, about 20% and about 10%, about 20% and about 15%, about 15% and about 1%, about 15% and about 5%, about 15% and about 10%, about 10% and about 1%, or about 10% and about 5% relative to [CC] and [CM].
  • the number (percentage) of HM is between about 39% and about 30%, about 30% and about 20%, about 20% and about 10%, about 10% and about 5%, and about 5% and about 1% relative to [CC] and [CM]. In some aspects, the number (percentage) of HM is about 39%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% relative to [CC] and [CM]. In some aspects, the number of HM is expressed as the percentage of [HM] relative to [CC] and [CM]. [0137] In some aspects, the cationic carrier unit of the present disclosure interacts with a nucleotide payload having about 100 to about 1000 nucleotides in length.
  • the nucleotide payload having about 100 to about 1000 nucleotides in length encodes one or more proteins or fragments thereof, e.g,. PAPD5/7 or any fragments therof.
  • the carrier unit complexed the nucleotide payload having about 100 to about 1000 nucleotides in length forms a micelle.
  • the cationic carrier unit of the present disclosure interacts with a nucleotide payload having about 1000 to about 2000 nucleotides in length.
  • the nucleotide payload having about 1000 to about 2000 nucleotides in length encodes one or more proteins or fragments thereof, e.g., G protein or any fragments therof.
  • the carrier unit complexed the nucleotide payload having about 1000 to about 2000 nucleotides in length forms a micelle.
  • the cationic carrier unit of the present disclosure interacts with a nucleotide payload having about 2000 to about 3000 nucleotides in length.
  • the nucleotide payload having about 2000 to about 3000 nucleotides in length encodes one or more proteins or fragments thereof, e.g., Gephyrin or any fragments therof.
  • the carrier unit complexed the nucleotide payload having about 2000 to about 3000 nucleotides in length forms a micelle.
  • the cationic carrier unit of the present disclosure interacts with a nucleotide payload having about 3000 to about 4000 nucleotides in length.
  • the nucleotide payload having about 3000 to about 4000 nucleotides in length encodes one or more proteins or fragments thereof, e.g., MDA5 (IFIH1) or any fragments therof.
  • the carrier unit complexed the nucleotide payload having about 3000 to about 4000 nucleotides in length forms a micelle.
  • the vitamin B3 unit are introduced into the side chains of the HM moiety, e.g., by a coupling reaction between NH2 groups in the lysines and COOH groups of vitamin B3, in the presence of suitable conjugation reagents, for example, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS).
  • suitable conjugation reagents for example, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS).
  • EDC 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide
  • NHS N-hydroxy succinimide
  • the present disclosure provides complexes comprising a carrier unit (e.g., a cationic carrier unit unit) of the present disclosure non- covalently attached to a payload (e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), wherein the carrier unit and the payload interact electrostatically.
  • a carrier unit e.g., a cationic carrier unit unit
  • a payload e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • the present disclosure provides conjugates comprising a carrier unit (e.g., a cationic carrier unit unit) of the present disclosure covalently attached to a payload (e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), wherein the carrier unit and the payload interact electrostatically.
  • a carrier unit e.g., a cationic carrier unit unit
  • a payload e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • the carrier unit and the payload can be linked via a cleavable linker.
  • the carrier unit and the payload in addition to interacting electrostatically, can interact covalently (e.g., after electrostatic interaction the carrier unit and the payload can be "locked" via a disulfide bond or a cleavable bond).
  • the cationic carrier unit comprises a water-soluble polymer comprising a PEG with about 120 to about 130 units, a cationic carrier moiety comprising a polylysine with about 20 to about 60 lysine units, a crosslinking moiety comprising about 3 to about 40 lysine-thiol units, and a hydrophobic moiety comprising about 1 to about 20 lysines linked to a vitamin B3 units.
  • the cationic carrier unit is associated with a negatively charged payload (e.g., a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), which interacts with the cationic carrier unit via at least one ionic bond (i.e., via electrostatic interaction) with the cationic carrier moiety of the cationic carrier unit.
  • a negatively charged payload e.g., a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • ionic bond i.e., via electrostatic interaction
  • the specific components of the cationic carrier units of the present disclosure are disclosed in detail below. a. Water-soluble biopolymer
  • the cationic carrier units of the present disclosure comprise at least one water-soluble biopolymer.
  • water-soluble biopolymer refers to a biocompatible, biologically inert, non-immunogenic, non-toxic, and hydrophilic polymer, e.g., PEG.
  • the water-soluble polymer comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • POZ polyoxazolines
  • the water-soluble biopolymer is linear, branched, or dendritic.
  • the water-soluble biopolymer comprises polyethylene glycol ("PEG"), polyglycerol (“PG”), or poly(propylene glycol) (“PPG”).
  • PPG is less toxic than PEG, so many biological products are now produced in PPG instead of PEG.
  • the water-soluble biopolymer comprises a PEG characterized by a formula R 3 -(O-CH2-CH2)n- or R 3 -(0-CH2-CH2)n-O- with R 3 being hydrogen, methyl or ethyl and n having a value from 2 to 200.
  • the PEG has the formula (Formula III) wherein n is 1 to 1000.
  • the n of the PEG has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
  • n is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about
  • n is between about 50 and about 100, between about 100 and about 150, between about 150 and about 200, between about 200 and about 250, between about 250 and about 300, between about 300 and about 350, between about 350 and about 400, between about 400 and about 450, between about 450 and about 500, between about 500 and about 550, between about 550 and about 600, between about 600 and about 650, between about 650 and about 700, between about 700 and about 750, between about 750 and about 800, between about 800 and about 850, between about 850 and about 900, between about 900 and about 950, or between about 950 and about 1000.
  • n is at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about
  • n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 85 to about 95, about 95 to about 105, about 105 to about 115, about 115 to about 125, about 125 to about 135, about 135 to about 145, about 145 to about 155, about 155 to about 165, about 80 to about 100, about 100 to about 120, about 120 to about 140, about 140 to about 160, about 85 to about 105, about 105 to about 125, about 125 to about 145, or about 145 to about 165. [0155] In some aspects, n is about 100 to about 150.
  • n is about 100 to about 140. In some aspects, n is about 100 to about 130. In some aspects, n is about 110 to about 150. In some aspects, n is about 110 to about 140. In some aspects, n is about 110 to about 130. In some aspects, n is about 110 to about 120. In some aspects, n is about 120 to about 150. In some aspects, n is about 120 to about 140. In some aspects, n is about 120 to about 130. In some aspects, n is about 130 to about 150. In some aspects, n is about 130 to about 140. [0156] Thus, is some aspects, the PEG is a branched PEG. Branched PEGs have three to ten PEG chains emanating from a central core group.
  • the PEG moiety is a monodisperse polyethylene glycol.
  • a monodisperse polyethylene glycol is a PEG that has a single, defined chain length and molecular weight. mdPEGs are typically generated by separation from the polymerization mixture by chromatography.
  • a monodisperse PEG moiety is assigned the abbreviation mdPEG.
  • the PEG is a Star PEG. Star PEGs have 10 to 100 PEG chains emanating from a central core group.
  • the PEG is a Comb PEGs. Comb PEGs have multiple PEG chains normally grafted onto a polymer backbone.
  • the PEG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PEG is PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900, PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 1600, PEG 1700, PEG 1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900, PEG 3000, PEG 3100, PEG 3200 , PEG 3300 , PEG 3400 , PEG 3500 , PEG 3600 , PEG 3700 , PEG 3800 , PEG 3900 , PEG 4000 , PEG 3100, PEG 3
  • the PEG is PEG5000. In some aspects, the PEG is PEG6000. In some aspects, the PEG is PEG4000. [0160] In some aspects, the PEG is monodisperse, e.g., mPEG 100 , mPEG 200, mPEG 300, mPEG400, mPEG500, mPEG600, mPEG700, mPEG800, mPEG900, mPEG1000, mPEG1100, mPEG1200, mPEG1300, mPEG1400, mPEG1500, mPEG1600, mPEG1700, mPEG1800, mPEG1900, mPEG2000, mPEG2100, mPEG 2200, mPEG 2300, mPEG 2400, mPEG 2500, mPEG 1600, mPEG 1700, mPEG 1800, mPEG 1900, mPEG 2000,
  • the mPEG is mPEG5000. In some aspects, the mPEG is mPEG6000. In some aspects, the mPEG is mPEG4000.
  • the water-soluble biopolymer moiety is a polyglycerol (PG) described by the formula ((R 3 —O—(CH 2 —CHOH—CH 2 O)n—) with R 3 being hydrogen, methyl or ethyl, and n having a value from 3 to 200.
  • PG polyglycerol
  • the water-soluble biopolymer moiety is a branched polyglycerol described by the formula (R 3 —O—(CH 2 —CHOR 5 —CH 2 —O) n —) with R 5 being hydrogen or a linear glycerol chain described by the formula (R 3 —O—(CH 2 — CHOH—CH2—O)n—) and R 3 being hydrogen, methyl or ethyl.
  • the water-soluble biopolymer moiety is a hyperbranched polyglycerol described by the formula (R 3 —O—(CH2— CHOR 5 —CH 2 —O) n —) with R 5 being hydrogen or a glycerol chain described by the formula (R 3 — O—(CH2—CHOR 6 —CH2—O)n—), with R 6 being hydrogen or a glycerol chain described by the formula (R 3 —O—(CH2—CHOR 7 —CH2—O)n—), with R 7 being hydrogen or a linear glycerol chain described by the formula (R 3 —O—(CH 2 —CHOH—CH 2 —O) n —) and R 3 being hydrogen, methyl or ethyl.
  • the PG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PG is PG100, PG200, PG300, PG400, PG500, PG600, PG700, PG800, PG 900, PG 1000, PG 1100, PG 1200, PG 1300, PG 1400, PG 1500, PG 1600, PG 1700, PG 1800, PG 1900, PG 2000, PG 2100, PG2200, PG2300, PG2400, PG2500, PG1600, PG1700, PG1800, PG1900, PG2000, PG2100, PG2200, PG2300, PG2400, PG2500, PG2600, PG2700, PG2800, PG2900, PG3000, PG3100, PG3200, PG3300, PG3400, PG3500, PG3600, PG3700, PG 3800 , PG 3900 , PG 4000 , PG 4100, PG 4000 , PG
  • the PG is PG 5000 . In some aspects, the PG is PG6000. In some aspects, the PG is PG4000. [0164] In some aspects, the PG is monodisperse, e.g., mPG100, mPG200, mPG300, mPG400, mPG 500, mPG 600, mPG 700, mPG 800, mPG 900, mPG 1000, mPG 1100, mPG 1200, mPG 1300, mPG 1400, mPG 1500, mPG1600, mPG1700, mPG1800, mPG1900, mPG2000, mPG2100, mPG2200, mPG2300, mPG2400, mPG2500, mPG1600, mPG1700, mPG1800, mPG1900, mPG2000, mPG2100, mPG2200, mPG2300, mPG2400, mPG2500,
  • the water-soluble biopolymer comprises poly(propylene glycol) ("PPG").
  • PPG is characterized by the following formula, with n having a value from 1 to 1000.
  • the n of the PPG has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
  • n of the PPG is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about
  • the n of the PPG is between about 50 and about 100, between about 100 and about 150, between about 150 and about 200, between about 200 and about 250, between about 250 and about 300, between about 300 and about 350, between about 350 and about 400, between about 400 and about 450, between about 450 and about 500, between about 500 and about 550, between about 550 and about 600, between about 600 and about 650, between about 650 and about 700, between about 700 and about 750, between about 750 and about 800, between about 800 and about 850, between about 850 and about 900, between about 900 and about 950, or between about 950 and about 1000.
  • the n of the PPG is at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about
  • the n of the PPG is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 85 to about 95, about 95 to about 105, about 105 to about 115, about 115 to about 125, about 125 to about 135, about 135 to about 145, about 145 to about 155, about 155 to about 165, about 80 to about 100, about 100 to about 120, about 120 to about 140, about 140 to about 160, about 85 to about 105, about 105 to about 125, about 125 to about 145, or about 145 to about 165.
  • the PPG is a branched PPG. Branched PPGs have three to ten PPG chains emanating from a central core group.
  • the PPG moiety is a monodisperse polyethylene glycol.
  • a monodisperse polyethylene glycol is a PPG that has a single, defined chain length and molecular weight. mdPEGs are typically generated by separation from the polymerization mixture by chromatography.
  • mdPPG is assigned the abbreviation mdPPG.
  • the PPG is a Star PPG.
  • Star PPGs have 10 to 100 PPG chains emanating from a central core group.
  • the PPG is a Comb PPGs.
  • Comb PPGs have multiple PPG chains normally grafted onto a polymer backbone.
  • the PPG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PPG is PPG 100 , PPG 200, PPG 300, PPG 400, PPG 500, PPG 600, PPG 700, PPG 800, PPG 900, PPG 1000, PPG 1100, PPG 1200, PPG 1300, PPG 1400, PPG 1500, PPG 1600, PPG 1700, PPG 1800, PPG1900, PPG2000, PPG2100, PPG2200, PPG2300, PPG2400, PPG2500, PPG1600, PPG1700, PPG1800, PPG1900, PPG 2000, PPG 2100, PPG 2200, PPG 2300, PPG 2400, PPG 2500, PPG 2600, PPG 2700, PPG 2800, PPG 2900, PPG 3000, PPG 3100, PPG 3200 , PPG 3300 , PPG 3400 , PPG 3500 , PPG 3600 , PPG 3700 , PPG 3800 , PPG 3900 , PPG 4000, PPG 3100, P
  • the PPG is PPG5000. In some aspects, the PPG is PPG6000. In some aspects, the PPG is PPG4000. [0175] In some aspects, the PPG is monodisperse, e.g., mPPG 100 , mPPG 200, mPPG 300, mPPG 400, mPPG 500, mPPG 600, mPPG 700, mPPG 800, mPPG 900, mPPG 1000, mPPG 1100, mPPG 1200, mPPG1300, mPPG1400, mPPG1500, mPPG1600, mPPG1700, mPPG1800, mPPG1900, mPPG2000, mPPG2100, mPPG 2200, mPPG 2300, mPPG 2400, mPPG 2500, mPPG 1600, mPPG 1700, mPPG 1800, mPPG 1900, mPPG 2000
  • the mPPG is mPPG 5000 . In some aspects, the mPPG is mPPG 6000 . In some aspects, the mPPG is mPPG4000.
  • the cationic carrier units of the present disclosure comprise at least one cationic carrier moiety.
  • the term "cationic carrier" refers to a moiety or portion of a cationic carrier unit of the present disclosure comprising a plurality of positive charges that can interact and bind electrostatically an anionic payload (or an anionic carrier attached to a payload).
  • the number of positive charges or positively charged groups on the cationic carrier is similar to the number of negative charges or negatively charged groups on the anionic payload (or an anionic carrier attached to a payload).
  • the cationic carrier comprises a biopolymer, e.g., a peptide (e.g., a polylysine).
  • the cationic carrier comprises one or more basic amino acids (e.g., lysine, arginine, histidine, or a combination thereof).
  • the cationic carrier comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55,
  • the cationic carrier unit comprises at least about 40 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 45 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 50 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 55 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 60 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 65 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises at least about 70 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 75 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 80 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises about 30 to about 1000, about 30 to about 900, about 30 to about 800, about 30 to about 700, about 30 to about 600, about 30 to about 500, about 30 to about 400, about 30 to about 300, about 30 to about 200, about 30 to about 100, about 40 to about 1000, about 40 to about 900, about 40 to about 800, about 40 to about 700, about 40 to about 600, about 40 to about 500, about 40 to about 400, about 40 to about 300, about 40 to about 200, or about 40 to about 100 basic amino acids, e.g., lysines.
  • basic amino acids e.g., lysines.
  • the basic amino acids e.g., lysines
  • the basic amino acids are not modified such that they possess –NH3+(e.g., positive charge).
  • the cationic carrier unit comprises about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 70 to about 80, about 75 to about 85, about 65 to about 75, about 65 to about 80, about 60 to about 85, or about 40 to about 500 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises about 100 to about 1000, about 100 to about 900, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 200, about 200 to about 1000, about 200 to about 900, about 200 to about 800, about 200 to about 700, about 200 to about 600, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 300 to about 1000, about 300 to about 900, about 300 to about 800, about 300 to about 700, about 300 to about 600, about 300 to about 500, about 300 to about 400, about 400 to about 1000, about 400 to about 900, about 400 to about 800, about 400 to about 700, about 400 to about 600, about 400 to about 500, about 500 to about 1000, about 500 to about 600, about 600 to about 1000, about 600 to about 900, about 600 to about 800, about 600 to about 700, about 400 to about 600, about 400 to about 500, about 500 to about 1000, about 500 to about 600, about 600
  • the number of basic amino acids can be adjusted based on the length of the anionic payload. For example, an anionic payload with a longer sequence can be paired with higher number of basic amino acids, e.g., lysines.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit can be calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20.
  • N/P ratio molar ratio of protonated amine in polymer to phosphate in an anionic payload
  • the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA is between about 1 to about 20, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 1 to about 10, e.g., about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 1 to about 2.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 3 to about 4.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 2 to about 3.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 4 to about 5. In some aspects, the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 5 to about 6.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 6 to about 7.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 7 to about 8.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 8 to about 9.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 9 to about 10.
  • a role of the cationic carrier moiety is to neutralize negative charges on the payload (e.g., negative changes in the phosphate backbone of an mRNA) via electrostatic interaction, in some aspects (e.g., when the payload is a nucleic acid such as an antimir), the length of the cationic carrier, number of positively charged groups on the cationic carrier, and distribution and orientation of charges present on the cationic carrier will depend on the length and charge distribution on the payload molecule.
  • the cationic carrier comprises between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, between about 25 and about 30, between about 30 and about 35, between about 35 and about 40, between about 40 and about 45, between about 45 and about 50, between about 50 and about 55, between about 55 and about 60, between about 60 and about 65, between about and about 70, between about 70 and about 75, or between about 75 and about 80 basic amino acids.
  • the positively charged carrier comprises between 30 and about 50 basic amino acids.
  • the positively charged carrier comprises between 70 and about 80 basic amino acids.
  • the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the basic amino acid is a D-amino acid. In some aspects, the basic amino acid is an L-amino acid. In some aspects, the positively charged carrier comprises D- amino acids and L-amino acids. In some aspects, the basic amino comprises at least one unnatural amino acid or a derivative thereof.
  • the basic amino acid is arginine, lysine, histidine, L-4-aminomethyl-phenylalanine, L-4-guanidine-phenylalanine, L-4-aminomethyl-N- isopropyl-phenylalanine, L-3-pyridyl-alanine, L-trans-4-aminomethylcyclohexyl-alanine, L-4- piperidinyl-alanine, L-4-aminocyclohexyl-alanine, 4-guanidinobutyric acid, L-2-amino-3- guanidinopropionic acid, DL-5-hydroxylysine, pyrrolysine, 5-hydroxy-L-lysine, methyllysine, hypusine, or any combination thereof.
  • the positively charged carrier comprises about 40 lysines. In a particular aspect, the positively charged carrier comprises about 50 lysines. In a particular aspect, the positively charged carrier comprises about 60 lysines. In a particular aspect, the positively charged carrier comprises about 70 lysines. In a particular aspect, the positively charged carrier comprises about 80 lysines.
  • the cationic carrier comprises a polymer or copolymer comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at last 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63,
  • the cationic carrier comprises a polymer or copolymer comprising between about 5 and about 10 cationic groups, between about 10 and about 15 cationic groups, between about 15 and about 20 cationic groups, between about 20 and about 25 cationic groups, between about 25 and about 30 cationic groups, between about 30 and about 35 cationic groups, between about 35 and about 40 cationic groups, between about 40 and about 45 cationic groups, between about 45 and about 50 cationic groups, between about 50 and about 55 cationic groups, between about 55 and about 60 cationic groups, between about 60 and about 65 cationic groups, between about 65 and about 70 cationic groups, between about 70 and about 75 cationic groups, or between about 45 and about 50 cationic groups (e.g., amino groups).
  • amino groups e.g., amino groups
  • the cationic carrier comprises a polymer or copolymer comprising between 30 and about 50 cationic groups (e.g., amino groups). In some specific aspects, the cationic carrier comprises a polymer or copolymer comprising between 70 and about 80 cationic groups (e.g., amino groups). In some aspects, the polymer or copolymer is an acrylate, a polyalcohol, or a polysaccharide. [0187] In some aspects, the cationic carrier moiety binds to a single payload molecule. In other aspects, a cationic carrier moiety can bind to multiple payload molecules, which may be identical or different.
  • the positive charges of the cationic carrier moiety and negative charges of a nucleic acid payload are at an ionic ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1 about 7:1, about 6:1 about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
  • the negative charges of a nucleic acid payload and the positive charges of the cationic carrier moiety are at an ionic ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1 about 7:1, about 6:1 about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
  • the anionic payload comprises a nucleotide sequence having about 10 to about 1000 (e.g., about 100 to about 1000) in length, wherein the N/P ratio of the the cationic carrier moiety and the anionic payload is about 2 to about 10, e.g., about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, e.g., e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • an N/P ratio of the cationic carrier moiety and the anionic payload of about 10 to about 1000 nucleotides in length is between about 1 and about 10, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • the anionic payload comprises a nucleotide sequence having about 1000 to about 2000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 12, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.
  • the N/P ratio of the cationic carrier moiety and the anionic payload is between about 4 and about 7, e.g., about 4, about 5, about 6, or about 7.
  • the anionic payload comprises a nucleotide sequence having about 2000 to about 3000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 16, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16.
  • the N/P ratio of the cationic carrier moiety and the anionic payload is between about 6 and about 9, e.g., about 6, about 7, about 8, or about 9.
  • the anionic payload comprises a nucleotide sequence having about 3000 to about 4000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 20, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In some aspects, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is between about 7 and about 10, e.g., about 7, about 8, about 9, or about 10. [0193] In some aspects, the cationic carrier moiety has a free terminus wherein the end group is a reactive group.
  • the cationic carrier moiety has a free terminus (e.g., the C-terminus in a poly-lysine cationic carrier moiety) wherein the end group is an amino (-NH 2 ) group. In some aspects, the cationic carrier moiety has a free terminus wherein the end group is an sulfhydryl group. In some apects, the reactive group of the cationic carrier moiety is attached to a hydrophobic moiety, e.g., a vitamin B3 hydrophobic moiety.
  • the cationic carrier units of the present disclosure comprise at least one crosslinking moiety.
  • crosslinking moiety refers to a moiety or portion of a polymer block comprising a plurality of agents that are capable of forming crosslinks.
  • the number of agents that are capable of forming crosslinks comprises an amino acid with a side chain of a crosslinking agent.
  • the CM comprises a biopolymer, e.g., a peptide (e.g., a polylysine) linked to a crosslinking agent.
  • the crosslinking moiety comprises one or more amino acids (e.g., lysine, arginine, histidine, or a combination thereof).
  • the crosslinking moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 amino acids, e.g., lysines, arginines, or combinations thereof, each
  • the crosslinking moiety comprises at least about 10 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 11 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 12 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 13 amino acids, e.g., lysines, each of which is linked to a crosslinking agent.
  • the crosslinking moiety comprises at least about 14 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 15 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 16 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 17 amino acids, e.g., lysines, each of which is linked to a crosslinking agent.
  • the crosslinking moiety comprises at least about 18 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 19 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 20 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. [0197] In some aspects, a crosslinking agent is a thiol. In some aspects, a crosslinking agent is a thiol derivative. d.
  • the cationic carrier units of the present disclosure comprise at least one hydrophobic moiety.
  • hydrophobic moiety refers to a molecular entity that can, e.g., (i) complement the therapeutic or prophylactic activity of the payload, (ii) modulate the therapeutic or prophylactic activity of the payload, (iii) function as a therapeutic and/or prophylactic agent in the target tissue or target cells, (iv) facilitate the transport of the cationic carrier unit across a physiological barrier, e.g., the BBB and/or the plasma membrane, (v) improve the homeostasis of the target tissue or target cell, (vi) contribute positively charges groups to the cationic carried moiety, or (vii) any combination thereof.
  • the hydrophobic moiety is capable of modulating, e.g., an immune response, an inflammatory response, or a tissue microenvironment.
  • a hydrophobic moiety capable of modulating an immune response can comprise, e.g., tyrosine or dopamine. Tyrosine can be transformed into L-DOPA, and then be converted to dopamine via 2-step enzymatic reaction. Normally, dopamine levels are low in the Parkinson’s disease patients. Therefore, in some aspects, tyrosine is a hydrophobic moiety in cationic carrier units used for the treatment of Parkinson’s disease.
  • cationic carrier units of the present disclosure used for the treatment of disease or conditions related to low serotonin levels comprise tryptophan as a hydrophobic moiety.
  • a hydrophobic moiety can modulate a tumor microenvironment in a subject with a tumor, for example, by inhibiting or reducing hypoxia in the tumor microenvironment.
  • the hydrophobic moiety comprises, e.g., an amino acid linked to an imidazole derivative, a vitamin, or any combination thereof.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to an imidazole derivative comprising: (Formula VI), wherein each of G1 and G2 is independently H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring, and wherein n is 1-10.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to nitroimidazole. Nitroimidazoles function as antibiotics. Nitroheterocycles in nitroimidazoles can be reductively activated in hypoxic cells, and then undergo redox recycling or decompose to cytotoxic products.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, nitroimidazole, or any combination thereof.
  • the hydrophobic moiety comprises (Formula VII), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • the hydrophobic moiety is capable of inhibiting or reducing an inflammatory response.
  • the hydrophobic moiety is an amino acid (e.g., lysine) linked to a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: (Formula VIII), wherein each of Y1 and Y2 is C, N, O, or S, and wherein n is 1 or 2.
  • the vitamin is selected from the group consisting of vitamin A (retinol), vitamin B1 (Thiamine Chloride), vitamin B2 (Riboflavin), vitamin B3 (Niacinamide), vitamin B6 (Pyridoxal), vitamin B7 (Biotin), vitamin B9 (Folic acid), vitamin B12 (Cobalamin), vitamin C (Ascorbic acid), vitamin D2, vitamin D3, vitamin E (Tocopherol), vitamin M, vitamin H, a derivative thereof, and any combination thereof.
  • the vitamin is vitamin B3 (also known as niacin or nicotinic acid).
  • the hydrophobic moiety comprises at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 1 amino acid (e.g., lysine), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 2 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 3 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 4 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 5 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 6 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 2 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 3 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 4 amino acids
  • the hydrophobic moiety comprises about 7 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 8 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 9 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 11 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 12 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 13 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 14 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 15 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 16 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 12 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 13 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 14 amino acids
  • the hydrophobic moiety comprises about 17 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 18 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 19 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3..
  • the hydrophobic moiety comprises from about 1 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 5 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 10 to about 15 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 15 to about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 1 to about 20 vitamin amino acids (e.g., lysines), each of which is linked to B3, about 1 to about 15 vitamin amino acids (e.g., lysines), each of which is linked to B3, about 1 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 1 to about 5 amino acids (e.g., lysines), each of which is linked to vitamin B3 [0212] Niacin is a precursor of the coenzymes nicotinamide
  • NAD converts to NADP by phosphorylation in the presence of the enzyme NAD+ kinase.
  • NADP and NAD are coenzymes for many dehydrogenases, participating in many hydrogen transfer processes.
  • NAD is important in catabolism of fat, carbohydrate, protein, and alcohol, as well as cell signaling and DNA repair, and NADP mostly in anabolism reactions such as fatty acid and cholesterol synthesis.
  • High-energy requirements (brain) or high turnover rate (gut, skin) organs are usually the most susceptible to their deficiency.
  • Niacin produces marked anti-inflammatory effects in a variety of tissues – including the brain, gastrointestinal tract, skin, and vascular tissue – through the activation of NIACR1.
  • the cationic carrier unit comprises a targeting moiety, which is linked to the water-soluble polymer optionally via a linker.
  • targeting moiety refers to a biorecognition molecule that binds to a specific biological substance or site.
  • the targeting moiety is specific for a certain target molecule (e.g., a ligand targeting a receptor, or an antibody targeting a surface protein), tissue (e.g., a molecule that would preferentially carry the micelle to a specific organ or tissue, e.g., liver, brain, or endothelium), or facilitate transport through a physiological barrier (e.g., a peptide or other molecule that may facilitate transport across the brain blood barrier or plasma membrane).
  • a target molecule e.g., a ligand targeting a receptor, or an antibody targeting a surface protein
  • tissue e.g., a molecule that would preferentially carry the micelle to a specific organ or tissue, e.g., liver, brain, or endothelium
  • a physiological barrier e.g., a peptide or other molecule that may facilitate transport across the brain
  • a targeting moiety can be coupled to a cationic carrier unit, and therefore, to the external surface of a micelle, whereas the micelle has the payload entrapped within its core.
  • the targeting moiety is a targeting moiety capable of targeting the micelle of the present disclosure to a tissue.
  • the tissue is liver, brain, kidney, lung, ovary, pancreas, thyroid, breast, stomach, or any combination thereof.
  • the tissue is cancer tissue, e.g., liver cancer, brain cancer, kidney cancer, lung cancer, ovary cancer, pancreas cancer, thyroid cancer, breast cancer, stomach cancer, or any combination thereof.
  • the tissue is liver.
  • the targeting moiety targeting liver is cholesterol.
  • the targeting moiety targeting liver is a ligand that binds an asialoglycoprotein receptor-targeting moiety.
  • the asialoglycoprotein receptor-targeting moiety comprises a GalNAc cluster.
  • the GalNAc cluster is a monovalent, divalent, trivalent, or tetravalent GalNAc cluster.
  • the tissue is pancreas.
  • the targeting moiety targeting pancreas comprises a ligand targeting ⁇ v ⁇ 3 integrin receptors on pancreatic cells.
  • the targeting moiety comprises an arginylglycylaspartic acid (RGD) peptide sequence (L- Arginyl-Glycyl-L-Aspartic acid; Arg-Gly-Asp).
  • RGD arginylglycylaspartic acid
  • the tissue is a tissue in the central nervous system, e.g., neural tissue.
  • the targeting moiety targeting the central nervous system is capable being transported by Large-neutral Amino Acid Transporter 1 (LAT1).
  • LAT1 Large-neutral Amino Acid Transporter 1
  • LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. LAT1 can transport drugs such as L-dopa or gabapentin.
  • a targeting moiety comprises glucose, e.g., D-glucose, which can bind to Glucose transporter 1 (or GLUT1) and cross BBB.
  • GLUT1 also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), is a uniporter protein that in humans is encoded by the SLC2A1 gene. GLUT1 facilitates the transport of glucose across the plasma membranes of mammalian cells.
  • a targeting moiety comprises galactose, e.g., D-galactose, which can bind to GLUT1 transporter to cross BBB.
  • a targeting moiety comprises glutamic acid, which can bind to acetylcholinesterase inhibitor (AChEI) and/or EAATs inhibitors and cross BBB.
  • Acetylcholinesterase is the enzyme that is the primary member of the cholinesterase enzyme family.
  • an acetylcholinesterase inhibitor is the inhibitor that inhibits acetylcholinesterase from breaking down acetylcholine into choline and acetate, thereby increasing both the level and duration of action of the neurotransmitter acetylcholine in the central nervous system, autonomic ganglia and neuromuscular junctions, which are rich in acetylcholine receptors.
  • Acetylcholinesterase inhibitors are one of two types of cholinesterase inhibitors; the other being butyryl-cholinesterase inhibitors.
  • the tissue targeted by a targeting moiety is a skeletal muscle.
  • the targeting moiety targeting skeletal muscle is capable being transported by Large- neutral Amino Acid Transporter 1 (LAT1).
  • LAT1 Large- neutral Amino Acid Transporter 1
  • T-cells T-cells
  • cancer cells T-cells
  • brain endothelial cells LAT1 is consistently expressed at high levels in brain microvessel endothelial cells.
  • LAT1 Being a solute carrier located primarily in the BBB, targeting the micelles of the present disclosure to LAT1 allows delivery through the BBB.
  • the targeting moiety targeting a micelle of the present disclosure to the LAT1 transporter is an amino acid, e.g., a branched-chain or aromatic amino acid.
  • the amino acid is valine, leucine, and/or isoleucine.
  • the amino acid is tryptophan and/or tyrosine. In some aspects, the amino acid is tryptophan. In other aspects, the amino acid is tyrosine.
  • the targeting moiety is a LAT1 ligand selected from tryptophan, tyrosine, phenylalanine, tryptophan, methionine, thyroxine, melphalan, L-DOPA, gabapentin, 3,5- I-diiodotyrosine, 3-iodo-I-tyrosine, fenclonine, acivicin, leucine, BCH, methionine, histidine, valine, or any combination thereof.
  • a ligand functions as a type of targeting moiety defined as a selectively bindable material that has a selective (or specific), affinity for another substance.
  • the ligand is recognized and bound by a usually, but not necessarily, larger specific binding body or "binding partner,” or "receptor.”
  • binding partner or “receptor.”
  • ligands suitable for targeting are antigens, haptens, biotin, biotin derivatives, lectins, galactosamine and fucosylamine moieties, receptors, substrates, coenzymes and cofactors among others.
  • a ligand When applied to the micelles of the present disclosure a ligand includes an antigen or hapten that is capable of being bound by, or to, its corresponding antibody or fraction thereof.
  • viral antigens or hemagglutinins and neuraminidases and nucleocapsids including those from any DNA and RNA viruses, AIDS, HIV and hepatitis viruses, adenoviruses, alphaviruses, arenaviruses, coronaviruses, flaviviruses, herpesviruses, myxoviruses, oncornaviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, reoviruses, rhabdoviruses, rhinoviruses, togaviruses and viroids; any bacterial antigens including those of gram-negative and gram-positive bacteria, Acinetobacter, Achromobacter, Bacteroides, Clostridium, Chlamydia, enterobacteria, Haemophilus, Lactobacillus, Neisseria, Staphyloccus, or Strept
  • cancer and tumor antigens such as alpha-fetoproteins, prostate specific antigen (PSA) and CEA, cancer markers and oncoproteins, among others.
  • Other substances that can function as ligands for targeting a micelle of the present disclosure are certain vitamins (i.e. folic acid, B 12 ), steroids, prostaglandins, carbohydrates, lipids, antibiotics, drugs, digoxins, pesticides, narcotics, neuro-transmitters, and substances used or modified such that they function as ligands.
  • the targeting moiety comprises a protein or protein fragment (e.g., hormones, toxins), and synthetic or natural polypeptides with cell affinity.
  • Ligands also include various substances with selective affinity for ligators that are produced through recombinant DNA, genetic and molecular engineering. Except when stated otherwise, ligands of the instant disclosure also include ligands as defined in U.S. Pat. No. 3,817,837, which is herein incorporated by reference in its entirety.
  • a ligator functions as a type of targeting moiety defined for this disclosure as a specific binding body or "partner" or “receptor,” that is usually, but not necessarily, larger than the ligand it can bind to. For the purposes of this disclosure, it can be a specific substance or material or chemical or "reactant" that is capable of selective affinity binding with a specific ligand.
  • a ligator can be a protein such as an antibody, a nonprotein binding body, or a "specific reactor.”
  • a ligator includes an antibody, which is defined to include all classes of antibodies, monoclonal antibodies, chimeric antibodies, Fab fractions, fragments and derivatives thereof.
  • the term “antibody” encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain.
  • Antibody further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, scFab, (scFab) 2 , (scFv) 2 , Fab, Fab', and F(ab') 2 , F(ab1) 2 , Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides.
  • Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
  • the targeting moiety is an antibody or a molecule comprising an antigen binding fragment thereof.
  • the antibody is a nanobody.
  • the antibody is an ADC.
  • the terms "antibody-drug conjugate” and “ADC” are used interchangeably and refer to an antibody linked, e.g., covalently, to a therapeutic agent (sometimes referred to herein as agent, drug, or active pharmaceutical ingredient) or agents.
  • the targeting moiety is an antibody-drug conjugate. [0233] Under certain conditions, the instant disclosure is also applicable to using other substances as ligators.
  • ligators suitable for targeting include naturally occurring receptors, any hemagglutinins and cell membrane and nuclear derivatives that bind specifically to hormones, vitamins, drugs, antibiotics, cancer markers, genetic markers, viruses, and histocompatibility markers.
  • Another group of ligators includes any RNA and DNA binding substances such as polyethylenimine (PEI) and polypeptides or proteins such as histones and protamines.
  • Other ligators also include enzymes, especially cell surface enzymes such as neuraminidases, plasma proteins, avidins, streptavidins, chalones, cavitands, thyroglobulin, intrinsic factor, globulins, chelators, surfactants, organometallic substances, staphylococcal protein A, protein G, ribosomes, bacteriophages, cytochromes, lectins, certain resins, and organic polymers.
  • Targeting moieties also include various substances such as any proteins, protein fragments or polypeptides with affinity for the surface of any cells, tissues or microorganisms that are produced through recombinant DNA, genetic and molecular engineering.
  • the targeting moiety directs a micelle of the present disclosure to a specific tissue (i.e., liver tissue or brain tissue), to a specific type of cell (e.g., a certain type of cancer cells), or to a physiological compartment or physiological barrier (e.g., the BBB).
  • a cationic carrier unit disclosed herein can comprise, as shown, e.g., in FIG. 2, one or more linkers.
  • linker refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence), or a non-peptide linker for which its main function is to connect two moieties in a cationic carrier unit disclosed herein.
  • cationic carrier units of the present disclosure can comprise at least one linker connecting a tissue-specific targeting moiety (TM) with a water soluble polymer (WS), at least one linker connecting a water-soluble biopolymer (WP) with cationic carrier (CC) or a hydrophobic moiety (HM) or a crosslinking moiety (CM), at least one linker connecting a cationic carrier (CC) with a hydrophobic moiety (HM), or any combination thereof.
  • two or more linkers can be linked in tandem.
  • each of the linkers can be the same or different.
  • linkers provide flexibility to the cationic carrier unit.
  • Linkers are not typically cleaved; however, in certain aspects, such cleavage can be desirable. Accordingly, in some aspects a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the linker sequence. [0238] In one aspect, the linker is a peptide linker.
  • the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.
  • the peptide linker can comprise at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 amino acids.
  • the peptide linker can comprise at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, or at least about 1,000 amino acids.
  • the peptide linker can comprise between 1 and about 5 amino acids, between 1 and about 10 amino acids, between 1 and about 20 amino acids, between about 10 and about 50 amino acids, between about 50 and about 100 amino acids, between about 100 and about 200 amino acids, between about 200 and about 300 amino acids, between about 300 and about 400 amino acids, between about 400 and about 500 amino acids, between about 500 and about 600 amino acids, between about 600 and about 700 amino acids, between about 700 and about 800 amino acids, between about 800 and about 900 amino acids, or between about 900 and about 1000 amino acids.
  • Examples of peptide linkers are well known in the art. In some aspects, the linker is a glycine/serine linker.
  • the peptide linker is glycine/serine linker according to the formula [(Gly)n-Ser]m where n is any integer from 1 to 100 and m is any integer from 1 to 100.
  • the glycine/serine linker is according to the formula [(Gly)x-Sery]z (SEQ ID NO: 1) wherein x in an integer from 1 to 4, y is 0 or 1, and z is an integers from 1 to 50.
  • the peptide linker comprises the sequence Gn, where n can be an integer from 1 to 100.
  • the sequence of the peptide linker is GGGG (SEQ ID NO: 2).
  • the peptide linker can comprise the sequence (GlyAla)n (SEQ ID NO: 3), wherein n is an integer between 1 and 100. In other aspects, the peptide linker can comprise the sequence (GlyGlySer)n (SEQ ID NO: 4), wherein n is an integer between 1 and 100. [0244] In other aspects, the peptide linker comprises the sequence (GGGS)n (SEQ ID NO: 5). In still other aspects, the peptide linker comprises the sequence (GGS)n(GGGGS)n (SEQ ID NO: 6). In these instances, n can be an integer from 1-100.
  • n can be an integer from one to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 7), GGSGGSGGSGGSGGG (SEQ ID NO: 8), GGSGGSGGGGSGGGGS (SEQ ID NO: 9), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 10), or GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • the linker is a poly-G sequence (GGGG)n (SEQ ID NO: 12), where n can be an integer from 1-100.
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g., natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides that are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion).
  • the peptide linker can comprise non-naturally occurring amino acids.
  • the peptide linker can comprise naturally occurring amino acids occurring in a linear sequence that does not occur in nature. In still another aspect, the peptide linker can comprise a naturally occurring polypeptide sequence. [0247] In some aspects, the linker comprises a non-peptide linker. In other aspects, the linker consists of a non-peptide linker.
  • the non-peptide linker can be, e.g., maleimido caproyl (MC), maleimido propanoyl (MP), methoxyl polyethyleneglycol (MPEG), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N- succinimidyl(4-iodoacetyl)aminobenzonate (SIAB), succinimidyl 6-[3-(2-pyridyldithio)- propionamide]hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), etc.
  • MC maleimido caproy
  • Linkers can be introduced into polypeptide sequences using techniques known in the art (e.g., chemical conjugation, recombinant techniques, or peptide synthesis). Modifications can be confirmed by DNA sequence analysis. In some aspects, the linkers can be introduced using recombinant techniques. In other aspects, the linkers can be introduced using solid phase peptide synthesis. In certain aspects, a cationic carrier unit disclosed herein can contain simultaneously one or more linkers that have been introduced using recombinant techniques and one or more linkers that have been introduced using solid phase peptide synthesis or methods of chemical conjugation known in the art.
  • the linker comprises a cleavage site.
  • payload refers to a biologically active molecule, e.g., a therapeutic agent that can interactive by itself or via an adapter with a cationic carrier unit of the present disclosure, and be included within the core of a micelle of the present disclosure.
  • Other biologically active molecules are anti-viral drugs, nucleic acids and other anti-viral substances including those against any DNA and RNA viruses, AIDS, HIV and hepatitis viruses, adenoviruses, alphaviruses, arenaviruses, coronaviruses, flaviviruses, herpesviruses, myxoviruses, oncornaviruses, papovaviruses, paramyxoviruses, parvoviruses, picomaviruses, poxviruses, reoviruses, thabdoviruses, rhinoviruses, togaviruses and viriods; any anti-bacterial drugs, nucleic acids and other anti-bacterial substances including those against gram-negative and grampositive bacteria, Acinetobacter, Achromobacter, Bacteroides, Clostridium, Chlamydia, enterobacteria, Haemophilus, Lactobacillus, Neisseria, Staphyloccu
  • the biologically active molecule is a nucleic acid, e.g., an RNA or a DNA.
  • Nucleic acid active agents suitable for delivery using the micelles of the present disclosure include all types of RNA and all types of DNA.
  • the nucleic acid comprises mRNA, miRNA sponge, tough decoy miRNA (TD), cDNA, pDNA, PNA, BNA, aptamer, or any combination thereof.
  • the biologically active molecule e.g., anionic payload
  • the biologically active molecule (e.g., anionic payload) comprises a nucleotide sequence having less than about 3,500, less than about 3,000, less than about 2,500, less than about 2,000, less than about 1,500, less than about 1,000, less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 200, or less than about 150 in length.
  • the nucleic acids are phosphodiester nucleotides, and any nucleotides where the sugar-phosphate "backbone” has been derivatized or replaced with "backbone analogues” such as with phosphorothioate, phosphorodithioate, phosphoroamidate, alkyl phosphotriester, or methylphosphonate linkages.
  • the nucleic acids have non- phosphorous backbone analogues such as sulfamate, 3'-thioformacetal, methylene (methylimino) (MMI), 3'-N-carbamate, or morpholino carbamate.
  • the anionic payload is a polynucleotide, also can be referred to as nucleotide or nucleic acid.
  • polynucleotide in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides.
  • Exemplary polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ - L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino- ⁇ -LNA having a 2’-amino functionalization) or hybrids thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the synthetic polynucleotide is a synthetic messenger RNA (mRNA).
  • mRNA messenger RNA
  • the term “messenger RNA” (mRNA) refers to any polynucleotide, which may be synthetic, which encodes a polypeptide, e.g., a G protein, and which is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.
  • the present disclosure expands the scope of functionality of traditional mRNA molecules by providing synthetic polynucleotides which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotides including, in some aspects, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a synthetic polynucleotide, primary construct or mmRNA without significant chemical modification to the nucleotides themselves.
  • the anionic payload can include untranslated regions. Untranslated regions (UTRs) of a gene are transcribed but not translated.
  • the 5’ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3’ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into the polynucleotides, primary constructs and/or mmRNA of the present invention to enhance the stability of the molecule.
  • the specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
  • any UTR from any gene may be incorporated into the respective first or second flanking region of the primary construct.
  • multiple wild-type UTRs of any known gene can be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type genes. These UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’ UTR can be inverted, shortened, lengthened, made chimeric with one or more other 5’ UTRs or 3’ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3’ or 5’ UTR can be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’) comprise a variant UTR.
  • the nucleotide also include a poly A tail, a miRNA binding site, an AU element, or any combination thereof.
  • an anionic payload having about 100 to about 1000 nucleotides in length can be any protein or fragments thereof having about 30 amino acids or less in length.
  • an anionic payload having about 100 to about 1000 nucleotides in length encodes PAPD5/7 (0.8 kb), or any fragments thereof.
  • an anionic payload having about 1000 to about 2000 nucleotides in length can be any protein or fragments thereof having about 660 amino acids or less in length.
  • an anionic payload having about 1000 to about 2000 nucleotides in length can encode G protein (1.5 kb).
  • an anionic payload having about 2000 to about 3000 nucleotides in length can be any protein or fragments thereof having about 1000 amino acids or less in length.
  • an anionic payload having about 2000 to about 3000 nucleotides in length can encode Gephyrin, protein anchor (2.2 kb).
  • an anionic payload having about 3000 to about 4000 nucleotides in length can be any protein or fragments thereof having about 1330 amino acids or less in length.
  • an anionic payload having about 3000 to about 4000 nucleotides in length can encode MDA5 (IFIH1) (3.0 kb). i.
  • a polynucleotide of the present disclosure comprises at least one chemically modified nucleoside and/or nucleotide.
  • the polynucleotides of the present disclosure are chemically modified, the polynucleotides can be referred to as "modified polynucleotides.”
  • a "nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase").
  • a “nucleotide” refers to a nucleoside including a phosphate group.
  • Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • the modified polynucleotides disclosed herein can comprise various distinct modifications.
  • the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide can exhibit one or more desirable properties, e.g., improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to the target gene or protein, reduced non-specific binding to other gene or other molecules, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present disclosure is chemically modified.
  • the terms "chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population, including, but not limited to, its nucleobase, sugar, backbone, or any combination thereof.
  • a polynucleotide of the present disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation
  • the polynucleotide of the present disclosure e.g., an mRNA
  • Modified nucleotide base pairing encompasses not only the standard adenine- thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleobase inosine and adenine, cytosine, or uracil.
  • TD TD
  • the polynucleotide (e.g., an mRNA) includes a combination of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more) modified nucleobases.
  • the chemical modification is at nucleobases in a polynucleotide of the present disclosure (e.g., an mRNA).
  • the at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine ( ⁇ ), 2-thiouridine (s2U), 1-methyl- pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), or 5-methoxy-uridine (mo5U)), a modified cytosine (e.g., 5-methyl-cytidine (m5C)) a modified adenosine (e.g, 1-methyl-adenosine (m1A), N6-methyl-adenosine (m6A), or 2-methyl-adenine (m2A)), a modified guanosine (e.g., 7-methyl- guanosine (m7G) or 1-methyl-guanosine (m1G)), or a combination thereof.
  • a modified uridine e.g., pseudouridine ( ⁇ ), 2-thiouridine (s2U), 1-methyl- pseudouridine (m1 ⁇ ), 1-e
  • the polynucleotide of the present disclosure is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with the same type of base modification, e.g., 5-methyl-cytidine (m5C), meaning that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C).
  • m5C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified nucleoside such as any of those set forth above. 2.
  • the payload can comprise a "polynucleotide of the present disclosure" (for example comprising an mRNA), wherein the polynucleotide includes any useful modification to the linkages between the nucleosides.
  • Such linkages, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3'-alkylene phosphonates, 3'-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH 2 - O-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -, -CH 2 -NH-CH 2 -, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, morpholino linkages, -N(CH 3 )-CH 2 -CH 2 -, oli
  • the presence of a backbone linkage disclosed above increase the stability (e.g., thermal stability) and/or resistance to degradation (e.g., enzyme degradation) of a polynucleotide of the present disclosure (e.g., an mRNA).
  • the stability and/or resistance to degradation increases by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 100% in the modified polynucleotide compared to a corresponding polynucleotide without the modification (reference or control polynucleotide) [0278]
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 backbone linkages in a polynucleotide of the present disclosure are modified (e.g., phosphorothioate).
  • the backbone comprises linkages selected from the group consisting of phosphodiester linkage, phosphotriesters linkage, methylphosphonate linkage, phosphoramidate linkage, phosphorothioate linkage, and combinations thereof. 3.
  • the modified nucleosides and nucleotides which can be incorporated into a polynucleotide of the present disclosure can be modified on the sugar of the nucleic acid.
  • the payload comprises a nucleic acid, wherein the nucleic comprises at least one nucleoside analog (e.g., a nucleoside with a sugar modification).
  • affinity-enhancing nucleotide analogues in the polynucleotide can allow the length of polynucleotide to be reduced, and also may reduce the upper limit of the size a polynucleotide before non-specific or aberrant binding takes place.
  • sugar modifications e.g., LNA
  • nucleotide units in a polynucleotide of the present disclosure are sugar modified (e.g., LNA).
  • sugar modified e.g., LNA
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.
  • GAA glyco
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • the 2’ hydroxyl group (OH) of ribose can be modified or replaced with a number of different substituents.
  • Exemplary substitutions at the 2’-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl-C 1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to
  • nucleoside analogues present in a polynucleotide of the present disclosure comprise, e.g., 2’-O-alkyl-RNA units, 2’-OMe-RNA units, 2’-O-alkyl- SNA, 2’-amino-DNA units, 2’-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2’-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2’MOE units, or any combination thereof.
  • ANA arabino nucleic acid
  • INA intercalating nucleic acid
  • the LNA is, e.g., oxy-LNA (such as beta-D-oxy-LNA, or alpha-L-oxy-LNA), amino-LNA (such as beta-D-amino-LNA or alpha-L-amino-LNA), thio-LNA (such as beta-D-thio0-LNA or alpha-L-thio-LNA), ENA (such a beta-D-ENA or alpha-L-ENA), or any combination thereof.
  • oxy-LNA such as beta-D-oxy-LNA, or alpha-L-oxy-LNA
  • amino-LNA such as beta-D-amino-LNA or alpha-L-amino-LNA
  • thio-LNA such as beta-D-thio0-LNA or alpha-L-thio-LNA
  • ENA such a beta-D-ENA or alpha-L-ENA
  • nucleoside analogs present in a polynucleotide of the present disclosure comprise Locked Nucleic Acid (LNA); 2'-0-alkyl-RNA; 2'-amino-DNA; 2'-fluoro- DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-0-methyl nucleic acid (2'-OMe), 2'-0- methoxyethyl nucleic acid (2'-MOE), or any combination thereof.
  • LNA Locked Nucleic Acid
  • 2'-amino-DNA 2'-fluoro- DNA
  • arabino nucleic acid ANA
  • 2'-fluoro-ANA hexitol nucleic acid
  • INA intercalating nucleic acid
  • cEt constrained ethyl nucleoside
  • the present disclosure also provides micelles comprising the cationic carrier units of the present disclosure.
  • the micelles of the present disclosure comprise cationic carriers unit of the present disclosure and negatively charged payload, wherein the negatively charged payload and the cationic carrier unit are associate with each other.
  • the association is comprises a covalent bond.
  • the association does not comprise a covalent bond.
  • the association is via an ionic bond, i.e., via electrostatic interaction.
  • the negatively charged payload (e.g., a DNA and/or RNA) is not conjugated to the cationic carrier unit by a covalent bond and/or the negatively charged payload interacts with the cationic carrier moiety of the cationic carrier unit only via an ionic interaction.
  • the cationic carrier units and micelles of the present disclosure protect the payload (e.g., a DNA and/or RNA) from degradation (e.g., by a DNase and/or an RNase).
  • the cationic carrier unit is capable of protecting the payload through electrostatic interaction.
  • the micelle sequesters the payload to the core of the micelle, i.e., out of the reach of DNases and/or an RNases.
  • the protection of the payload from circulating enzymes can increase the half-life of the negatively charged payload (e.g., a DNA and/or RNA) compared to the free payload.
  • encapsulation of the payload in a micelle of the present disclosure can increase the plasma half-life of the payload at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11- fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold , at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least about 30-fold compared to the free payload.
  • the positive charge of the cationic carrier unit, and in particular the charge of the cationic carrier moiety is sufficient to form a micelle when mixed with a negatively charged payload (e.g., a nucleic acid) in a solution, wherein the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
  • a negatively charged payload e.g., a nucleic acid
  • the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 1:20, about 1:19, about 1:18, about 1:17, about 1:16, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1;1.
  • the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 2:1.
  • the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 3:1. In some aspects, the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 4:1. In some aspects, the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 5:1.
  • the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 6:1. In some aspects, the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 7:1. In some aspects, the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 8:1.
  • the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 9:1. In some aspects, the overall ionic ratio between the cationic carrier unit, in particular its cationic carrier moiety, and the negatively charged payload (e.g., a nucleic acid) is about 10:1.
  • the anionic payload for the micelles comprises a nucleotide sequence having about 10 to about 1000 (e.g., about 100 to about 1000) in length, wherein the N/P ratio of the the cationic carrier unit and the anionic payload is about 2 to about 10, e.g., about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, e.g., e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • an N/P ratio of the cationic carrier unit and the anionic payload of about 10 to about 1000 nucleotides in length is between about 1 and about 10, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • the anionic payload for the micelles comprises a nucleotide sequence having about 1000 to about 2000 in length, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 3 to about 12, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.
  • the N/P ratio of the cationic carrier uit and the anionic payload is between about 4 and about 7, e.g., about 4, about 5, about 6, or about 7.
  • the anionic payload for the micelles comprises a nucleotide sequence having about 2000 to about 3000 in length, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 3 to about 16, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16.
  • the N/P ratio of the cationic carrier unit and the anionic payload is between about 6 and about 9, e.g., about 6, about 7, about 8, or about 9.
  • the anionic payload for the micelles comprises a nucleotide sequence having about 3000 to about 4000 in length, wherein the N/P ratio of the cationic carrier unit and the anionic payload is about 3 to about 20, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In some aspects, wherein the N/P ratio of the cationic carrier unit and the anionic payload is between about 7 and about 10, e.g., about 7, about 8, about 9, or about 10.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 7 and about 10. In some aspects, the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein the N/P ratio of the cationic carrier unit and the anionic payload in the solution is between about 7 and about 8 or about 8 and about 9.
  • the anionic payload and the cationic carrier unit are capable of forming a micelle when mixed together in a solution, wherein N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 7, about 8, about 9, or about 10.
  • N/P ratio of the cationic carrier unit and the anionic payload in the solution is about 7, about 8, about 9, or about 10.
  • the complexes formed between the cationic carrier units of the present disclosure and payload e.g., an mRNA
  • a micelle is a water soluble or colloidal structure or aggregate composed of one or more amphiphilic molecules.
  • Amphiphilic molecules are those that contain at least one hydrophilic (polar) moiety and at least one hydrophobic (nonpolar) moiety.
  • “Classic micelles” have a single, central and primarily hydrophobic zone or “core” surrounded by a hydrophilic layer or “shell.” In aqueous solution, the micelle forms an aggregate with the hydrophilic "head” regions of the amphiphilic molecule in contact with the surrounding solvent, sequestering the hydrophobic single-tail regions of the amphiphilic molecule in the micelle core.
  • Micelles are approximately spherical in shape. Other shapes, e.g., ellipsoids, cylinders, rod-like structures, or polymersomes are also possible.
  • the shape and size, and therefore loading capacity, of the micelles disclosed can be modified by altering the ratio between water-soluble biopolymer (e.g., PEG) and cationic carrier (e.g., poly lysine).
  • the carrier units can organize as small particles, small micelles, micelles, rod-like structures, or polymersomes.
  • the term "micelles of the present disclosure” encompasses not only classic micelles but also small particles, small micelles, micelles, rod-like structures, or polymersomes.
  • the micelles of the present disclosure can be composed of either a single monomolecular polymer containing hydrophobic and hydrophilic moieties or an aggregate mixture containing many amphiphilic (i.e.
  • Micelles of the present disclosure can range in size from 5 to about 2000 nanometers. In some aspects, the diameter of the micelle is between about 10 nm and about 200 nm.
  • the diameter of the micelle is between about 1nm and about 100nm, between about 10nm and about 100nm, between about 10nm and about 90nm, between about 10nm and about 80nm, between about 10nm and about 70nm, between about 20nm and about 100nm, between about 20nm and about 90nm, between about 20nm and about 80nm, between about 20nm and about 70nm, between about 30nm and about 100nm, between about 30nm and about 90nm, between about 30nm and about 80nm, between about 30nm and about 70nm, between about 40nm and about 100nm, between about 40nm and about 90nm, between about 40nm and about 80nm, or between about 40nm and about 70nm.
  • the diameter of the micelles of the present disclosure is between about 30 nm and about 60 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 15 nm and about 90 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 15 nm and about 80 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 15 nm and about 70 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 15 nm and about 60 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 15 nm and about 50 nm.
  • the diameter of the micelles of the present disclosure is between about 20 nm and about 60 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 20 nm and about 50 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 20 nm and about 40 nm. In some aspects, the diameter of the micelles of the present disclosure is between about 25 nm and about 35 nm. In some aspects, the diameter of the micelles of the present disclosure is about 32 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 100 nm and about 200 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 40 nm and about 50 nm.
  • the diameter of the micelles of the present disclosure is about about 50 nm and about 60 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 60 nm and about 70 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 70 nm and about 80 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 80 nm and about 90 nm. In some aspects, the diameter of the micelles of the present disclosure is about about 90 nm and about 100 nm. [0301] In some aspects, the micelles of the present disclosure comprise a single type of cationic carrier unit.
  • the micelles of the present disclosure comprise more than one type of cationic carrier unit (e.g., targeting different receptor on the surface of a target cell).
  • micelles of the present disclosure can comprise cationic carrier units with different targeting moieties, different cationic carrier moieties (e.g., to accommodate different payloads), and/or different hydrophobic and/or crosslinking units.
  • different types of cationic or anionic carrier unit can be combined together.
  • the micelle of the present disclosure can comprise a cationic (or an anionic) carrier unit linked to a targeting moiety and a cationic (or an anionic) carrier unit not linked to a targeting moiety.
  • a micelle comprises about 50 to about 200 cationic or anionic carrier units.
  • a micelle comprises about 50 to about 150, about 50 to about 140, about 50 to about 130, about 50 to about 120, about 50 to about 110, or about 50 to about 100 cationic or anionic carrier units.
  • a micelle comprises about 60 to about 200 cationic or anionic carrier units.
  • a micelle comprises about 60 to about 150, about 60 to about 140, about 60 to about 130, about 60 to about 120, about 60 to about 110, about 60 to about 100, about 60 to about 90, about 60 to about 80, or about 60 to about 70 cationic or anionic carrier units. In some aspects, a micelle comprises about 70 to about 200 cationic or anionic carrier units. In other aspects, a micelle comprises about 70 to about 150, about 70 to about 140, about 70 to about 130, about 70 to about 120, about 70 to about 110, about 70 to about 100, about 70 to about 90, or about 70 to about 80 cationic or anionic carrier units. In some aspects, a micelle comprises about 80 to about 200 cationic or anionic carrier units.
  • a micelle comprises about 80 to about 150, about 80 to about 140, about 80 to about 130, about 80 to about 120, about 80 to about 110, about 80 to about 100, or about 80 to about 90 cationic or anionic carrier units. In some aspects, a micelle comprises about 90 to about 200 cationic or anionic carrier units. In other aspects, a micelle comprises about 90 to about 150, about 90 to about 140, about 90 to about 130, about 90 to about 120, about 90 to about 110, or about 90 to about 100 cationic or anionic carrier units. In some aspects, a micelle comprises about 100 to about 200 cationic or anionic carrier units.
  • a micelle comprises about 100 to about 150, about 100 to about 140, about 100 to about 130, about 100 to about 120, about 100 to about 110, or about 100 to about 100 cationic or anionic carrier units.
  • the present disclosure also includes a micelle comprising (i) a nucleotide sequence (e.g., an mRNA having less than 4000 nucleotides in length) and (ii) a cationic carrier unit described herein.
  • the disclosure is directed to a micelle comprising (i) a nucleotide sequence, e.g., mRNA having less than 4000 nucleotides in length), and (ii) about 80 to about 120 (e.g., about 85 to about 115, about 90 to about 110, about 95 to about 105) cationic carrier units described herein, e.g., Schemas I-VI, Schemas I’-VI’, or a combination thereof (see FIG.2A-2E).
  • a nucleotide sequence e.g., mRNA having less than 4000 nucleotides in length
  • about 80 to about 120 e.g., about 85 to about 115, about 90 to about 110, about 95 to about 105
  • cationic carrier units described herein e.g., Schemas I-VI, Schemas I’-VI’, or a combination thereof (see FIG.2A-2E).
  • the micelle comprises (i) a nucleotide sequence, e.g., mRNA, and (ii) about 80 to about 120 (e.g., about 80, about 85, about 90, about 95, about 100, about 105, or about 110) of a cationic carrier unit described herein, e.g., optional [CC]-L1-[CM]-L2-[HM] (see FIG.2).
  • a cationic carrier unit described herein e.g., optional [CC]-L1-[CM]-L2-[HM] (see FIG.2).
  • the micelle comprises (i) a nucleotide sequence, e.g., mRNA), and (ii) about 60 to about 110, e.g., about 80, cationic carrier units, wherein (a) about 45 to about 90, e.g., about 80 of the cationic carrier units comprise [CC]-L1-[CM]-L2-[HM] and (b) about 45 to about 55, e.g., about 50 of the cationic carrier units comprise TM-[CC]-L1-[CM]-L2-[HM], wherein TM is phenyl alanine, WP is (PEG)5000, and CC is about 40 to about 50 lysines, e.g., about 45, about 46, about 47, about 48, about 49, or about 50 lysines, and wherein each of about 5 to about 15 of lysines, about 5 lysines, is fused to Vitamin B3 (nicotinamide).
  • a nucleotide sequence
  • the micelle can comprise a single payload (e.g., a single mRNA). In other aspects, the micelle can comprise more than one payload (e.g., multiple mRNA).
  • V. Methods of manufacture The present disclosure also provides methods of making the cationic carrier units and micelles of the present disclosure. In general, the present disclosure provides a method of preparing a cationic carrier unit of the present disclosure comprising synthesizing the cationic carrier unit as described, e.g., in the Examples section. As used herein, the term "synthesizing" refers the assembling the cationic carrier unit using methods known in the art.
  • protein components e.g., an antibody targeting moiety
  • each one of the components of the cationic carrier unit can be prepared using methods known in the art, e.g., recombinant protein production, solid phase peptide or nucleic acid synthesis, chemical synthesis, enzymatic synthesis, or any combination thereof, and the resulting component can be conjugated using chemical and/or enzymatic methods known in the art.
  • the cationic carrier units of the present disclosure can be purified to remove contaminants.
  • the cationic carrier unit comprises a uniform population of cationic carrier units.
  • the cationic carrier unit can comprise multiple species (e.g., some of them comprising a targeting moiety, and some comprising the remaining moieties but without a targeting moiety).
  • the manufacture of the cationic carrier units of the present disclosure comprise lyophilization or any other form of dry storage suitable for reconstitution.
  • the preparation of the cationic carrier unit in a dry form takes place after combination of the cationic carrier units with the payload (e.g., a nucleic acid).
  • the method of preparing a micelle of the present disclosure comprises mixing the cationic carrier unit with the negatively charged payload (e.g., a nucleic acid such as mRNA) at an ionic ratio of 1:1.
  • the cationic carrier unit and the negatively charged payload are combined in solution.
  • the resulting solution is lyophilized or dried.
  • the combination of the cationic carrier and the negative charged payload is conducted in dry form.
  • the ratio of number n of monomer units in the water-soluble polymer (e.g., PEG) to the number m of monomer units (e.g., lysines) in the cationic carrier moiety (e.g., poly lysine), wherein the number of units n or m in each case can be up to 1,000 units affects the size and shape of the resulting micelles.
  • the micelles obtained are classic micelles. If mB/(nA+mB) is above 0.5, the micelles obtained are rod like micelles or polymersomes. If mB/(nA+mB) is below 0.5, the micelles obtained are small micelles or small particles.
  • the micelles of the present disclosure can be generation using any of the techniques known in the art, for example, vortexing, extrusion, or sonication. The formation of micelles depends on applying conditions that are above the critical micelle concentration (CMC) of a solution comprising the cationic carrier units of the present disclosure.
  • CMC critical micelle concentration
  • the CMC of a solution comprising the cationic carriers of the present disclosure can be determined by any physical property (e.g., surface tension) that shows a distinct transition around the CMC.
  • the well-known Smith-Ewart theory predicts that the number of particles nucleated leading to the formation of micelles above the CMC is proportional to the surfactant (in the present disclosure, the cationic carrier units complexed or associated to the anionic payload) concentration to the 0.6 power. This is so because for a given surfactant the number of micelles formed generally increases with an increase in the surfactant concentration.
  • the micelles of the present disclosure can be purified, e.g., to remove contaminants and/or to generate an uniform population of micelles (e.g., micelles having the same size, or micelles having the same payload or the same targeting moiety).
  • the present disclosure also provides pharmaceutical compositions comprising cationic carrier units and/or micelles of the present disclosure (i.e., micelles comprising cationic carrier units of the present disclosure) that are suitable for administration to a subject.
  • micelles of the present disclosure can be homogeneous (i.e., all micelles comprises the same type of cationic carrier unit, with the same targeting moiety and the same payload).
  • the micelles can comprise multiple targeting moieties, multiple payloads, etc.
  • the pharmaceutical compositions generally comprise a cationic carrier unit and/or micelle of the present disclosure and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • suitable formulations of pharmaceutical compositions comprising micelles of the present disclosure See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.18th ed. (1990)).
  • the pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • the pharmaceutical composition comprises one or more micelles described herein.
  • the micelles described herein are co-administered with one or more additional therapeutic agents, in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprising the micelles described herein is administered prior to administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the micelles described herein is administered after the administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the micelles described herein is administered concurrently with the additional therapeutic agent(s).
  • the pharmaceutical carrier is added following micelle formation.
  • the pharmaceutical carrier is added before micelle formation.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyr
  • Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the cationic carrier units or micelles disclosed herein, use thereof in the compositions is contemplated.
  • Supplementary therapeutic agents can also be incorporated into the compositions of the present disclosure. Typically, a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • the micelles described herein can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular route or as inhalants.
  • the pharmaceutical composition micelles described herein is administered intravenously, e.g. by injection.
  • the micelles described herein can optionally be administered in combination with other therapeutic agents that are at least partly effective in treating the disease, disorder or condition for which the micelles described herein are intended.
  • Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition is generally sterile and fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be added to the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.
  • Pharmaceutical compositions of the present disclosure can be sterilized by conventional, well-known sterilization techniques. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • Sterile injectable solutions can be prepared by incorporating the micelles described herein in an effective amount and in an appropriate solvent with one or a combination of ingredients enumerated herein, as desired.
  • dispersions are prepared by incorporating the micelles described herein into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions comprising micelles described herein can be administered in the form of a depot injection or implant preparation that can be formulated in such a manner to permit a sustained or pulsatile release of the micelles described herein.
  • Systemic administration of compositions comprising micelles described herein can also be by transmucosal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished using, e.g., nasal sprays.
  • the pharmaceutical composition comprising micelles described herein is administered intravenously into a subject that would benefit from the pharmaceutical composition.
  • the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti et al., PNAS 105(46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or into the liver.
  • the pharmaceutical composition comprising micelles described herein is administered as a liquid suspension.
  • the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration.
  • the depot slowly releases the micelles described herein into circulation, or remains in depot form.
  • pharmaceutically acceptable compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
  • the pharmaceutically acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro- crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto.
  • the pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
  • the pharmaceutical compositions described herein comprise the micelles described herein and optionally a pharmaceutically active or therapeutic agent.
  • the therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.
  • Dosage forms are provided that comprise micelles described herein. In some aspects, the dosage form is formulated as a liquid suspension for intravenous injection.
  • the micelles disclosed herein or pharmaceutical composition comprising the micelles may be used concurrently with other drugs. To be specific, the micelles or pharmaceutical compositions of the present disclosure may be used together with medicaments such as hormonal therapeutic agents, chemotherapeutic agents, immunotherapeutic agents, medicaments inhibiting the action of cell growth factors or cell growth factor receptors and the like. VII.
  • the present disclosure also provides methods of treating a disease or condition in a subject in need thereof comprising administering a micelle of the present disclosure or a combination thereof to the subject, e.g., a mammal, such as human subject.
  • the present disclosure provides a method of treating a neurodegenerative disorder or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a micelle of the present disclosure, or a pharmaceutical composition of the present disclosure.
  • the micelles of the present disclosure can administered via intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the micelles of the present disclosure can be used concurrently with other medicaments or treatment suitable for the treatment of the diseases and conditions disclosed herein.
  • the present disclosure also provides methods to encapsulate a payload for delivery, comprising incorporating the payload, e.g., an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • the present disclosure also provides methods to increase the resistance of a payload to degradation (e.g., nuclease-mediated degradation), comprising incorporating the payload, e.g., an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • the present disclosure provides methods of crossing blood brain barrier (BBB) comprising administering the micelles disclosed herein, e.g., micelles comprising tryptophan and/or tyrosine as a targeting moiety.
  • BBB blood brain barrier
  • a micelle of the present disclosure loaded with mRNA can be targeted to a BBB receptor, e.g., LAT1, as disclosed above.
  • encapsulation of the payload in a micelle of the present disclosure can increase the resistance of the payload to degradation at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the free payload (i.e., not in a micelle, e.g., free in solution).
  • encapsulation of the payload in a micelle of the present disclosure can increase the resistance of the payload to degradation at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold , at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least about 30-fold compared to the free payload.
  • the present disclosure also provides methods to increase the stability of a payload during administration (e.g., while in the subject’s bloodstream) comprising incorporating the payload, e.g., an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • a payload e.g., an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • encapsulation of the payload in a micelle of the present disclosure can increase the stability (e.g., increase the resistance to nucleases) of the payload at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the free payload.
  • stability e.g., increase the resistance to nucleases
  • encapsulation of the payload in a micelle of the present disclosure can increase the stability (e.g., increase the resistance to nucleases) of the payload at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11- fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold , at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least about 30-fold compared to the free payload.
  • the present disclosure also provides methods to increase a payload’s plasma half- life comprising incorporating the payload, e.g., an anionic payload such as a nucleic acid (e.g., an mRNA) into a micelle of the present disclosure.
  • an anionic payload such as a nucleic acid (e.g., an mRNA)
  • encapsulation of the payload in a micelle of the present disclosure can increase the plasma half-life of the payload at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 1100%, at least about 1200%, at least about 1300%, at least about 1400%, at least about 1500%, at least about 1600%, at least about 1700%, at least about 1800%, at least about 1900%, or at least about 2000%,
  • encapsulation of the payload in a micelle of the present disclosure can increase the plasma half-life of the payload at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold , at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least about 30-fold compared to the free payload.
  • encapsulation of a payload in a micelle of the present disclosure can increase the permeation, delivery, transit, or transport of the payload through a physiological barrier, e.g., the BBB or the plasma membrane, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the free payload.
  • a physiological barrier e.g., the BBB or the plasma membrane
  • encapsulation of a payload in a micelle of the present disclosure can increase the permeation, delivery, transit, or transport of the payload through a physiological barrier, e.g., the BBB or the plasma membrane, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold , at least about 27-fold, at least about 28-fold, at least about 29-fold, or at least
  • the micelles of the present disclosure can be used to target stem cells, e.g., to deliver therapeutic molecules (e.g., therapeutic polynucleotides) or gene therapy components.
  • the micelles of the present disclosure can be used to treat cancer.
  • micelles of the present disclosure can target a marker specific for a certain type of cancer, e.g., a glioma, breast cancer, pancreatic cancer, liver cancer, skin cancer, or cervical cancer, and carry as payload a therapeutic molecule (e.g., a therapeutic polynucleotide, a peptide, or a small molecule).
  • the micelles of the present disclosure can be used to treat pancreatic cancer.
  • the targeting moiety directing the micelles of the present disclosure to pancreatic tissues is a cyclic RGD peptide. In other aspects, the targeting moiety directing the micelles of the present disclosure to pancreatic tissues is a biomarker predominantly or exclusively expressed on the surface of normal or cancerous pancreatic cells.
  • Kits [0350] The present disclosure also provides kits, or products of manufacture, comprising a cationic carrier unit, a micelle, or a pharmaceutical composition of the present disclosure and optionally instructions for use. In some aspects, the kit or product of manufacture comprises a cationic carrier unit, a micelle, or a pharmaceutical composition of the present disclosure in one or more containers.
  • the kit or product of manufacture comprises a cationic carrier unit, a micelle, or a pharmaceutical composition of the present disclosure and a brochure.
  • the kit or product of manufacture comprises a cationic carrier unit, a micelle, or a pharmaceutical composition of the present disclosure and instructions for use.
  • a cationic carrier unit, a micelle, or a pharmaceutical composition of the present disclosure, or combinations thereof can be readily incorporated into one of the established kit formats which are well known in the art.
  • the kit or product of manufacture comprises a cationic carrier unit of the present disclosure in dry form in a container (e.g., a glass vial), and optionally a vial with a solvent suitable to hydrate the dry the cationic carrier unit, and optionally instructions for the hydration of the cationic carrier unit and the formation of micelles.
  • a container e.g., a glass vial
  • the kit or product of manufacture further comprises at least one additional container (e.g., a glass vial) with the micelle’s anionic payload (e.g., an mRNA).
  • the kit or product of manufacture comprises a cationic carrier unit of the present disclosure in a dry form and the micelle’s anionic payload also in dry form in the same container, or in different containers.
  • the kit or product of manufacture comprises a cationic carrier unit of the present disclosure in solution and the micelle’s anionic payload also in solution in the same container, or in different containers.
  • the kit or product of manufacture comprises a micelle of the present disclosure in solution, and instructions for use.
  • the kit or product of manufacture comprises a micelle of the present disclosure in dry form, and instructions for use (e.g., instructions for reconstitution and administration).
  • the crude material was purified by flash column (EA in hexane 10%). Next, the resulting product was dissolved in Tetrahydrofuran (7.0 ml) and 6.0 M HCl (7.0 ml), and heated at 65 °C for 16 hrs. Afterwards, the dioxane was removed and the product was extracted using EA. Then, an aqueous NaOH (1.0 M) solution was added to the mixture until the pH value become 7. The reactant was concentrated by evaporator and centrifuged at 12,000 rpm at 0°C. The precipitate was washed with deionized water and lyophilized prior to use.
  • Lys(TFA)-NCA solution was dropped into the N3-PEG solution by micro syringe needle and the reaction mixture were stirred at 37 °C for 3 days.
  • the reaction bottles were purged with Ar and vacuum. All reactions were conducted under Ar atmosphere.
  • the mixture was precipitated into an excess amount of diethyl ether. Then, the mixture was filtered and white powder was obtained after drying in vacuo.
  • N3-PEG-PLL 500 mg was dissolved in methanol (60 mL), and 1N NaOH (6 mL) was dropped into the polymer solution with stirring. The mixture was maintained for 1 day with stirring at 37°C.
  • EDC (205.2 mg, 1 equiv. to NH2 of PEG-PLL) was added into nicotinic acid solution and NHS (123.2 mg, 1 equiv. to NH2 of PEG-PLL) stepwise added into the mixture. After 30 min of post incubation at room temperature, the reaction mixture was added into the N3-PEG-PLL(NH 2 ) solution. [0364] The reaction mixture was maintained at 37 °C for 16 hours with stirring. Secondly, 3,3’-dithiodipropionic acid (65.6 mg, 0.17 equiv. to NH2 of PEG-PLL), EDC (41.8 mg, 0.25 equiv.
  • FIG.2C A schematic of (PEG5K- PLL80(Nic19/SH23) is shown in FIG.2C.
  • FIG.2D A schematic of (PEG 5K - PLL80(Nic32/SH16) is shown in FIG.2D.
  • N 3 -PEG-PLL(Nic/ss) (30 mg, 3.2 ⁇ mol) and alkyne modified phenyl alanine (1.31 mg, 6.4 ⁇ mol) were dissolved in deionized water.
  • CuSO4•H2O (0.172 mg, 0.69 ⁇ mol) and ascorbic acid (0.3 mg, 1.7 ⁇ mol) were added into the mixture solution.
  • Example 2 Polyion Complex (PIC) micelle preparation [0373] Once the cationic carrier units of the present disclosure were generated as described in Example 1, micelles were produced. The micelles described in the present example comprised cationic carrier units combined with mRNA payloads with different lengths. [0374] (a) mRNA 800 nucleotides with Compound B: Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 5 /SH 35 ) and mRNA. PEG 5K - PLL 80 (Nic 5 /SH 35 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 1.26 mg/mL.
  • an mRNA solution (0.5 ⁇ M) in RNase free water was mixed with the polymer solution at a 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 3.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles. Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • SLI scattering light intensity
  • mRNA 800 nucleotides with Compound C Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 19 /SH 23 ) and mRNA. PEG 5K - PLL80(Nic19/SH23) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 1.32 mg/mL. Then, an mRNA solution (0.38 ⁇ M) in RNase free water was mixed with the polymer solution at a 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine- in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 4.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.25 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 800 nucleotides with Compound D Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG5K-PLL80(Nic32/SH16) and mRNA. PEG5K- PLL 80 (Nic 32 /SH 16 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 1.59 mg/mL. Then, an mRNA solution (0.3 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine- in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 5.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.2 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 1,800 nucleotides with Compound B Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 5 /SH 35 ) and mRNA. PEG 5K - PLL 80 (Nic 5 /SH 35 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 2.73 mg/mL. Then, an mRNA solution (0.25 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 6.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.17 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 1,800 nucleotides with Compound C Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 19 /SH 23 ) and mRNA. PEG 5K - PLL80(Nic19/SH23) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 2.85 mg/mL. Then, an mRNA solution (0.25 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 6.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.17 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 1,800 nucleotides with Compound D Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG5K-PLL80(Nic32/SH16) and mRNA. PEG5K- PLL 80 (Nic 32 /SH 16 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 3.44 mg/mL. Then, an mRNA solution (0.21 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine- in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 7.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.14 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 3,800 nucleotides with Compound B Nano-sized PIC micelle were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 5 /SH 35 ) and mRNA. PEG 5K - PLL 80 (Nic 5 /SH 35 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 6.03 mg/mL. Then, an mRNA solution (0.21 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 7.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.14 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 3,800 nucleotides with Compound C Nano-sized PIC micelle were prepared by mixing of MeO- or Phe-PEG 5K -PLL 80 (Nic 19 /SH 23 ) and mRNA. PEG 5K - PLL80(Nic19/SH23) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 6.3 mg/mL. Then, an mRNA solution (0.19 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 8.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.13 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • mRNA 3,800 nucleotides with Compound D Nano-sized PIC micelles were prepared by mixing of MeO- or Phe-PEG5K-PLL80(Nic32/SH16) and mRNA. PEG5K- PLL 80 (Nic 32 /SH 16 ) was dissolved in 200 mM DTT (in 10 mM HEPES buffer) at 7.59 mg/mL. Then, an mRNA solution (0.15 ⁇ M) in RNase free water was mixed with the polymer solution at 2:1 (v/v) ratio of mRNA to polymer.
  • the mixing ratios of polymer to mRNA were determined by optimization of the micelle-forming ratio between amine-in-polymer (N) and phosphate-in-mRNA (P). The optimal N to P ratio was 10.0.
  • the mixture of polymer and mRNA was vigorously mixed for 3 min by multi-vortex at 3000 rpm, and kept at room temperature for 30 min to stabilize the micelles.
  • Particle size distribution and scattering light intensity (SLI) were measured by Zeta-sizer with 634 nm wavelength.
  • the resulting micelles (0.1 ⁇ M of mRNA Conc.) were stored at 4 oC prior to use.
  • Example 3 In vitro mRNA expression [0383]
  • Cell line and culture The human embryonic kidney cell line (HEK-293T) and human lung cancer cell line (A549) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, LM 001-05) and RPMI 1640 (Welgene, LM 011-01) medium supplemented with 10% FBS (Gibco 26140-079) and penicillin streptomycin (Gibco 15140-122). Cells were seeded into 6-well plates at a density of 1 ⁇ 10 6 cells/well. Cells were cultured for 24 h in a culture medium containing 2% FBS and 0.5% penicillin-streptomycin.
  • DMEM Dulbecco’s modified Eagle’s medium
  • RPMI 1640 Welgene, LM 011-01
  • FBS Gibco 26140-079
  • penicillin streptomycin Gabco 15140-122
  • mRNA-loaded micelles or Lipofectamine 2000/mRNA complexes as a positive control were transfected into the cells.
  • Transfection of the cell line Cells were seeded into 6-well plates at a density of 1 ⁇ 10 6 cells/well. Cells were cultured for 24 h in medium containing 2% FBS and 0.5% penicillin- streptomycin and were transfected with PBS, mRNA-loaded micelles, or Lipofectamine 2000/mRNA complexes (as positive controls).
  • the Lipofectamine 2000 transfection reagent (Invitrogen, 11668019) was prepared by diluting 10 ⁇ L of stock solution into 100 ⁇ L of Opti-MEM.
  • Lipofectamine 2000 was mixed with 5 ⁇ g of mRNA. The mixture was gently pipetted and incubated at room temperature for 15 min to form Lipofectamine/mRNA complexes.
  • mRNA micelles were diluted with Opti-MEM to adjust the concentration of mRNA so it was the same concentration an is the Lipofectamine/mRNA complexes.
  • 5 ug of mRNA containing Lipofectamine/mRNA complexes or mRNA micelle were added into the cells respectively. After 30 min of post incubation, the cells were harvested, and RNA was isolated.
  • RNA extraction and qRT-PCR Total RNA was isolated from the cells using TRIzol reagent (Thermofisher, 15596018) according to the manufacturer's protocol. 500 ng of RNA were reverse transcribed into cDNA using the TOPscriptTM RT DryMIX (dN6 plus) (Enzynomics, RT210) according to the manufacturer's protocol. RT-qPCR was performed with the resultant cDNA as a template using TOPreal qPCR 2x PreMIX (SYBR Green with low ROX) (Enzynomics, RT500M) according to the manufacturer's protocol in the Bio-Rad CFX96 cycler (Bio-Rad Laboratories, Inc.).
  • Example 4 LAT-1 expression level in vivo LAT-1 distribution in mouse tissues: The animals were anesthetized and muscles (Gastrocnemius; GAS, Quadriceps femoris; QF, Biceps femoris; BF, Tibialis anterior; TA) were collected. Homogenized tissues were lysed using RIPA lysis and extraction buffer containing protease inhibitors, and protein concentration was determined using a BCA protein assay kit. [0391] Equal amounts of total protein were resolved by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membranes electrophoretically.
  • Example 5 In vivo mRNA expression [0393] 5 ⁇ g of mRNA encoding firefly luciferase (Luc) was formulated with polymeric carrier, Lipofectamine 2000 (Themo Fisher Scientific), and in vivo-jetRNA (Polyplus) in a total volume of 50 ⁇ l. [0394] Luc mRNA was injected into BALB/c mice through intramuscular administration (bilateral). Mice were injected VivoGlo Luciferin (Promega) at a dose of 150mg/kg intraperitoneally.
  • Bioluminescence imaging was performed with an IVIS imaging system (PerkinElmer).
  • Results The luciferase signals of Luc-mRNA encapsulated in Lipofectamine2000 (Lipo+Luc) or in vivo jetRNA (jetRNA+Luc, not shown) started to be observed after approximately 6 hours post-injection. The level of the signals was reduced by the first day, and practically no signal remained after 2 or 3 days. FIG.13A.
  • the maximum luciferase signal in the mRNA-loaded micelle treated group was not higher than the signal observed using other delivery reagents, the respone over time was different.

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